Visual performance and/or macular pigmentation

ABSTRACT

Disclosed is a composition comprising MZ for use as a dietary supplement or food additive for oral consumption for improving the visual performance of a human subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.K. Patent Application No.1111624.1, filed Jul. 7, 2011; U.K. Patent Application No. 1111625.8,filed Jul. 7, 2011; U.K. Patent Application No. 1207922.4, filed May 5,2012; U.K. Patent Application No. 1207923.2, filed May 5, 2012; thecontents of which are incorporated herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for improvingvisual performance in a human subject, and to methods of making thecomposition.

BACKGROUND OF THE INVENTION

The central retina, known as the macula, is responsible for color andfine-detail vision. A pigment, composed of the carotenoids, lutein (L),zeaxanthin (Z), and meso-zeaxanthin (MZ), accumulates at the maculawhere it is known as macular pigment (MP). MP is a blue light filter anda powerful antioxidant, and is therefore believed to protect againstage-related macular degeneration (AMD), which is now the most commoncause of blind registration in the western world. Various scientistshave proposed that macular pigments may enhance visual performance (VP),but there does not appear to be any persuasive experimental evidencesupporting such hypotheses.

MZ-containing compositions have been disclosed as useful in thetreatment of age-related macular degeneration (AMD), see for exampleU.S. Pat. No. 6,329,432. Supplements containing each of L, Z and MZ areknown, and sold for the intended purpose of treating and/or preventingeye disorders such as AMD. One example of such a supplement is soldunder the trade mark MacuShield®, and contains the three MP carotenoidsL, Z and MZ in the amounts of 10 mg, 2 mg and 10 mg respectively, perdose. WO 03/063848 discloses the use of a compound, such as lutein,zeaxanthin, mesozeaxanthin or mixtures thereof, for the manufacture of acomposition for improving visual performance of a subject in conditionsof darkness. The document is, however, rather unusual in that it doesnot contain any experimental evidence or data to support the allegeduse. The person skilled in the art would therefore be rather skepticalof the disclosure and certainly could not derive any expectation ofsuccess therefrom.

EP 1 920 711 discloses a method of assessing visual performance which,in effect, involves measuring or determining the amount of macularpigment (such as lutein, zeaxanthin or mesozeaxanthin) present in thesubject's eye (i.e. measuring macular pigment optical density, MPOD). Ifthe level of MPOD is low, the document suggests administering acomposition comprising lutein and/or zeaxanthin, which is purported tolead to an improvement in visual performance. However, the document doesnot disclose any actual experimental data to show that improving thelevel of macular pigment can produce an improvement in visualperformance. Again therefore, the person skilled in the art would treatthe disclosure of the document with some caution and could not deriveany expectation of success therefrom.

SUMMARY OF THE INVENTION

“Dietary supplement” means an addition to the diet in a pill, capsule,tablet, powder or liquid form, which is not a natural or conventionalfood, and which effectively increases the function of tissues or organs,or increases the level or concentration of a substance in the body, orimproves performance of tissues or organs.

The inventors have discovered that consumption of a dietary supplementcontaining lutein alone has little effect in the MP of subjects whoexhibit an abnormally low concentration of MP in the central portion ofthe retina. In contrast, consumption of a dietary supplement comprisingMZ alone can return MP levels in the central portion of the retinasubstantially to normal, but has little effect on MP levels outside thecentral portion. Consumption of a combined supplement, containingrelatively high amounts of MZ, but also Z and L, cannot only normalizeMP levels in the central region of the retina, but also augment MPlevels outside the central region of the retina.

For present purposes, the ‘central region’ of the retina means thatcentral portion of the retina which has an eccentricity of 0.25° orless, as determined by optical coherence tomography (OCT) and/or fundusphotography.

In a first aspect the invention provides a composition comprising MZ foruse as a dietary supplement, food additive or the like for oralconsumption improving the visual performance of a human subject. Inpreferred embodiments, the subject is a subject without age-relatedmacular degeneration (AMD).

For the purposes of the present specification, a subject is consideredto be without AMD if they have a score of 1-3 in the AREDS (Age-RelatedEye Disease Study) 11-step maculopathy grading system (Klein et al.,1991 Ophthalmology 98, 1128-1134).

In a second aspect, the invention provides a method of improving thevisual performance of a human subject in need of such improvement, themethod comprising the step of administering to the subject an effectiveamount of a composition comprising MZ. As explained below, thecomposition will preferably also comprise lutein and/or zeaxanthin. Thecomposition will preferably be administered orally, typically as adietary supplement or food additive. In preferred embodiments the methodis performed on a subject without AMD.

An effective amount of the composition for a particular subject canreadily be determined by non-inventive routine trial and error, in viewof the guidance given in the present specification. Low doses can begiven initially and the dosage increased until an improvement in visualperformance is detected. The subject's visual performance can be testedin any of a number of convenient methods, as elaborated below.

For present purposes, MZ is understood to refer to the compound (trans,3R, 3′S meso)-zeaxanthin, having the structure shown in FIG. 1. Alsoincluded within the term “MZ” are esters of MZ, for example the acetate,laurate, myristate, palmitate, linoleate, linolenate and arachidonateesters, and esters with omega 3 fatty acids.

A human subject is considered not to be experiencing AMD if, followingexamination by a retinologist, there are no signs of any of thefollowing characteristics normally associated with AMD including: softdrusen, hyper- and/or hypo-pigmentary changes at the macula (early AMD),or geographic atrophy or choroidal neovascularisation (advanced AMD).

The composition will preferably comprise MZ at a concentration of atleast 0.001% w/w up to 20% w/w. In one embodiment, a preferredconcentration of MZ may be in the range 3-10% w/w. However, the personskilled in the art will appreciate that the precise concentration of MZin the composition of the invention is not critical: a beneficial effecton the visual performance of the subject can be obtained by consuminglarger doses of a composition comprising lower concentrations of MZ andvice versa. A typical effective average daily dose of MZ to be consumedby a normal human adult subject will typically be in the range 0.1 mg to100 mg per day, more conveniently in the range 1 to 50 mg per day, andpreferably in the range 5-25 mg per day.

The composition may conveniently be in unitary dosage form e.g. as atablet, capsule or the like. Conveniently, but not necessarily, thecomposition may be packaged in a foil blister pack, of the sort known tothose skilled in the art. Desirably one or two of the doses are takeneach day, the amount of MZ in the doses being adjusted accordingly.

The composition of the invention will desirably comprise not only MZ,but also lutein and/or zeaxanthin. Most preferably the composition willcomprise MZ, lutein and zeaxanthin, which may be collectively referredto as macular carotenoids. Conveniently, but not necessarily, MZ will bepresent in the composition at a greater concentration or the sameconcentration as lutein or zeaxanthin. The percentage of either MZ orlutein in the composition can range from 10% to 90% (of macularcarotenoid pigment present in the formulation). The percentage ofzeaxanthin can typically range from 5 to 45% (of macular carotenoidpigment in the formulation). A particularly favored composition has anMZ:lutein:zeaxanthin ratio of 10:10:2 (or 45%, 45%, 10%).

The three macular carotenoids may be combined or preferably manufacturedas such in single formulation. The composition of the invention may bein any formulation suitable for oral consumption by a human subject,including a tablet, capsule, gel, liquid, powder or the like. Themacular carotenoids may be granulated for example as microcapsulesbefore inclusion in the formulation. The composition may convenientlycomprise conventional diluents, especially vegetable oils such assunflower, safflower, corn oil and rape seed oils, excipients, bulkingagents and the like which are well known to those skilled in the art.Such substances include calcium and/or magnesium stearate, starch ormodified starch.

Other conventional formulating agents may be present in the composition,including any one or more of the following non-exclusive list: acidityregulators; anticaking agents (e.g. sodium aluminosilicate, calcium ormagnesium carbonate, calcium silicate, sodium or potassiumferrocyanide), antioxidants (e.g. vitamin E, vitamin C, polyphenols),colorings (e.g. artificial colorings such as FD&C Blue No. 1, Blue No.2, Green No. 3, Red No. 40, Red No. 3, Yellow No. 5 and Yellow No. 6;and natural colorings such as caramel, annatto, cochineal, betanin,turmeric, saffron, paprika etc.); color retention agents; emulsifiers;flavors; flavor enhancers; preservatives; stabilizers; sweeteners andthickeners.

The above-mentioned compositions containing MZ can be an added to apreparation containing essential vitamins and minerals; for example aone a day tablet/capsule containing all RDAs of the vitamins andminerals required by man; or dietary products which are fortified byvitamins and minerals; or together with omega 3 fatty acids.

Macular carotenoids containing MZ can be fed to hens and the eggstherefrom can provide an excellent source of MZ for human consumption

Visual Performance

Visual performance is a state, condition or parameter, not anabnormality or a disease. Thus there is a range of values in normalsubjects without the presence of any underlying retinal or maculardisease. However, like all other human conditions, improvements in VPare considered beneficial and desirable.

There are many different measures of “visual performance” known to thoseskilled in the art.

For present purposes, improving “visual performance” means producing adetectable improvement in one or more of the following in the subject:contrast sensitivity; visual acuity, preferably best corrected visualacuity; glare disability; discomfort glare; ocular straylight;photostress recovery; and S-cone sensitivity. Preferably the improvementin visual performance created by consumption of the composition of theinvention comprises an improvement in one or more of: contrastsensitivity, best corrected visual activity, or glare disability.

Preferably consumption of the composition of the parameters of visualperformance, more preferably in two or more, and most preferably adetectable improvement in three or more of the aforementioned visualperformance parameters.

The various parameters of visual performance listed above are describedin more detail below.

(i) Contrast Sensitivity Function

Contrast is the difference in visual properties that make an object (orits representation in an image) distinguishable from other objects andthe background. In visual perception of the real world, contrast isdetermined by the difference in the color and brightness of the objectand other objects within the same field of view. Contrast Sensitivity isa measure of a subject's sensitivity to changes in contrast; it is ameasure of how much contrast is required to accurately detect a targetas distinct from its background.

By altering the size (spatial frequency) of a target, and the luminanceof the background, it is possible to test Contrast Sensitivity function,which is very much reflective of real-world vision, where the mostimportant determinants of vision are contrast, size and luminance.Contrast Sensitivity function can be assessed using the FunctionalAcuity Contrast Test (FACT), which is designed to test contrastsensitivity at varying spatial frequency settings, as disclosed byLoughman et al., 2010 Vision Res. 50, 1249-1256). Letter ContrastSensitivity may be measured using the commercially available “ThomsonChart”.

(ii) Visual Acuity

Visual acuity is a simple and intuitive way of assessing visualperformance It is a useful measure of vision because it relates directlyto the need for spectacles (i.e. if an individual is long or shortsighted, the introduction of spectacle lenses typically creates apredictable improvement in visual acuity). Also, it tends to beadversely affected by ocular disease and therefore abnormal visualacuity can be a sign of developing abnormality.

Despite its widespread use and popularity, it is not the best techniquefor the assessment of vision because (a) it tends not to relate wellwith vision in conditions different to the brightly lit, high contrasttest environment, and (b) it only evaluates performance at the highspatial frequency (i.e. small letter size) end of the spectrum.

Typically best corrected visual acuity (“BCVA”) is assessed using a highcontrast (close to 100%, i.e. black letters on a white background)letter chart, after the subject's vision has been corrected withcorrective lenses to the best level possible. The subject's task is toread the smallest possible letter size they can recognize. The visualperformance is quantified using a standard notation (e.g. Snellennotation; where 20/20 or 6/6 vision is accepted as normal human vision).Improvements in BCVA imply a benefit in visual acuity in general.

(iii) Glare Disability

Glare disability is a term used to describe the degradation of visualperformance typically caused by loss of retinal image contrast. Glaredisability is often caused, for example, by surface light reflections,or bright light sources such as car headlights, and typically is aconsequence of increased forward light scatter within the eye. Newbi-xenon high intensity discharge (“HID”) car headlights contain more“blue” light and are often considered as a cause of additional glaredisability compared to older headlight sources.

This is of particular importance to macular pigment investigationsbecause of the optical filtration properties of macular pigment. Macularpigment acts as a short wavelength (blue) light filter. Itsprereceptoral and central location facilitate the optimization of visualperformance with respect to glare because intraocular forward lightscatter is short wavelength (blue) light dominated.

Glare disability can be assessed using the Functional Acuity ContrastTest (FACT), as disclosed by Loughman et. al., 2010 Vision Res. 50,1249-1256.

(iv) Discomfort Glare

Discomfort glare results in an instinctive desire to look away from abright light source or difficulty in seeing a task. It refers to thesensation one experiences when the overall illumination is too brighte.g. on a snow field under bright sun.

Macular pigment has the capacity to diminish the effects of discomfortglare because (a) it filters the blue component which contains mostenergy; less light and less energy therefore reach the photoreceptors toaffect performance, and (b) macular pigment also has dichroic propertieswhich means it has the capacity to filter plane polarized light. Planepolarized light is light reflected from a surface (e.g. snow coveredground, water etc) into the eye. It is unidirectional so the energy isconcentrated and therefore has increased effect on vision. This is whyskiers, anglers and the like wear polarized sunglasses to reduce suchdiscomfort glare.

Discomfort glare is assessed using a discomfort rating scale asdisclosed by Wenzel et al., 2006 Vision Res. 46, 4615-4622.

(v) Ocular Straylight

Ocular straylight is a parameter that is relatively new in clinicalpractice after being studied for many years in experimental settings. Itconcerns the part of the incident light that is scattered by the ocularmedia and does not participate in the normal image formation on theretina. Instead, this light creates a more or less homogeneous haze overthe retinal image. Several pathologies are known to increase retinalstraylight considerably, which may lead to symptoms such as loss ofcontrast sensitivity, disability glare, and halos. This will reduce apatient's quality of vision in everyday life, for example while drivingat night and recognizing a person against a light source, but has only avery limited effect on visual acuity as measured during an ophthalmicexamination.

As macular pigment absorbs the dominant short wave scattered component,it has the capacity to significantly reduce the amount of ocularstraylight, and therefore further enrich visual performance particularlyunder circumstances of glare.

Ocular stray light is assessed using the Oculus C-Quant as disclosed byvan Bree et al., 2011 Ophthalmology 118, 945-953.

(vi) Photostress Recovery

Photostress Recovery testing is a method of assessing visual performanceby timing the recovery of visual function after adaptation to an intenselight source. The test involves exposing the macula to a light sourcebright enough to bleach a significant proportion of the visual pigments.Return of normal retinal function and sensitivity depends on theregeneration of the visual pigments. The test essentially provides anindirect assessment of macular function.

Photostress recovery is assessed using a macular automated photostresstest using the Humphrey Perimeter as disclosed by Loughman et. al., 2010Vision Res. 50, 1249-1256.

(vii) S-Cone Sensitivity

S-cones are the “blue” sensitive cones i.e. their peak sensitivity is toshort wavelengths. Typically, a person with high levels of macularpigment would be expected to demonstrate low S-cone sensitivity, as themacular pigment is minimizing the amount of blue light striking thephotoreceptors. Combining a test of S cone sensitivity with aphotostress test can provide information on the direct effects ofmacular pigment on the actual sensitivity of those cones most affectedby glare.

S-cone sensitivity is assessed using the short-wavelength automatedperimetry program (SWAP) on the Humphrey Perimeter as described by(Davison et. al., Optom. Vis. Sci. 2011 vol. 88).

(viii) Assessment of VP by Questionnaire

Another method of testing for improvement in visual performance is theuse of a questionnaire to score the subject's own assessment of theirvisual performance. In preferred embodiments of the invention therefore,a detectable improvement in visual performance is determined by anincreased score in a subjective assessment questionnaire following asuitable period of weeks or months of consumption of the composition, ascompared to a control assessment questionnaire completed prior tocommencing consumption of the composition.

A suitable questionnaire is disclosed by Charalampidou et al., Arch.Ophthalmol. 2011 (May 9^(th), Epublication ahead of print), in which isdescribed a 30-part, non validated, “Visual Function in Normals”questionnaire (VFNq30), which was designed to assess subjective visualperformance improvement. The design was based in part on apreviously-validated visual activities questionnaire (Sloane et al.,“The Visual Activities Questionnaire: Developing an instrument forassessing problems in everyday visual tasks. Technical Digest,Non-invasive Assessment of the Visual System, Topical Meeting of theOptical Society of America 1992), but adapted to suit a normal, youngand healthy population sample. This questionnaire allows the subject toquantify their visual performance using three separate metrics:situational analysis (SA) which requires the subject to rate theirvisual performance in specified daily life situations; comparativeanalysis (CA) which requires the subject to compare their perceivedvisual performance to that of their peers/family/friends; subjectsatisfaction score (SSS) which requires the subject to provide anoverall estimate of their perceived quality of vision. Each of the threemetrics above is computed to give a performance score for five differentfunctional aspects of their vision: acuity/spatial vision: glaredisability; light/dark adaptation; daily visual tasks; and colordiscrimination.

Time to Achieve an Improvement of VP

Obviously, one does not expect any measurable, discernible or detectableimprovement in the visual performance of a subject immediately afterconsuming the composition of the invention. The period of dietarysupplementation required to produce a measurable improvement in visualperformance will depend on several factors, including the average dailydose size of the macular carotenoids in the subject prior to commencingdietary supplementation, the subject's general health etc. Typically onewould expect to require dietary supplementation with the composition ofthe invention for at least 8 weeks, and more preferably at least 3 or 6months before measuring one or more visual performance parameters totest for any improvement therein.

The subject may need to consume the active composition of the inventionat least once a week, more normally at least 3 times a week, andpreferably daily.

Preferred Embodiments

In one embodiment of the invention, the composition may be consumed bysubjects who have a deficiency in the amount of macular pigment in thecentral portion of their macula. By way of explanation the inventorshave found that there exists a proportion of the population at large whomay not be experiencing AMD (as herein defined), but who possessstatistically significantly lower levels of macular pigment in thecentre of the macula as determined by customized heterochromatic flickerphotometry (cHFP) using the Macular Densitometer™. These subjects aredescribed as having an atypical macular pigment distribution, referredto as a “central dip”. Using this technique, MP may be measuredpsychophysically by HFP. HFP is based on the fact that MP absorbs bluelight. The subject may be asked to observe a target, within a testfield, which is alternating in square wave counterphase between blue(460 nm) and green light (550 nm), i.e. flickering. They must adjust theluminance of the blue light to achieve null flicker, in other words,until the target becomes steady. The ratio of the amount of blue lightrequired to achieve null flicker at the fovea may be compared to thatrequired in the para-fovea (where MP is presumed to be zero), thelogarithm of which is known as optical density. Using the Densitometer,MP can be measured at five points across the macula; 0.25°, 0.5°, 1°,1.75° and 7°. The principle of HFP remains the same for each target. Forthose retinal eccentricities outside the fovea, i.e. 0.5°, 1°, 1.75° and7°, the fixation point may be placed at the desired angular distancefrom a flickering disc. Three measurements may be taken at each loci andan average calculated. To minimize error in the HFP settings, care maybe taken to optimize the flicker rate for each subject, otherwise knownas critical flicker frequency (CFF). CFF is the frequency at which thesubject can no longer perceive flicker in a 0.5° target at 550 nm. TheCFF may be determined with a method of limits by which the flickeringfrequency is progressively decreased (or increased), until the subjectreports a change from fusion to flicker (or flicker to fusion). Subjectswith an atypical macular pigment distribution (“central dip”) may havean MPOD at 0.5° eccentricity which is greater than or equal to the MPODat 0.25° eccentricity.

In another embodiment, the composition may be consumed by subjects whohave statistically normal levels of macular pigment.

In another embodiment, the invention may provide a method of making acomposition for human consumption, the composition to be consumed by ahuman subject for the purpose of improving visual performance, themethod comprising the step of mixing an effective amount of MZ with anacceptable dietary diluent, excipient or carrier. The method mayadditionally comprise the addition of lutein and/or zeaxanthin to thediluent, excipient or carrier (or vice versa). Performance of the methodmay desirably result in manufacture of a composition having thepreferred features set forth above. The method may additionally comprisethe step of packaging the composition in a package together withinstructions for consumption of the composition to effect an improvementin visual performance. Conveniently, the composition may be packaged inunitary dose form e.g. as a plurality of tablets, capsules or pills,which may be packaged loose (e.g. in a tub) or may be packagedindividually (e.g. in a blister pack).

In one particular embodiment, the invention may provide a method ofimproving the visual performance of a human subject, the methodcomprising the steps of:

-   -   a) supplying a feed to egg-laying birds, such as hens or ducks,        which feed comprises MZ, so as to cause the birds to lay eggs        comprising MZ;    -   b) collecting said eggs, and supplying the eggs, or at least        part of the yolk thereof, in edible form to the subject.

Whole eggs may be provided raw for cooking by the subject. Alternativelythe eggs may be processed and at least part of the yolks thereofprovided to the subject, the MZ content of the eggs being concentratedin the yolk. Processing may involve, for example, shelling, cooking anddrying the eggs.

Typically the composition of the invention may be consumed at least oncea week, preferably at least twice a week, more preferably at least threetimes a week, and most preferably at least daily. In some embodimentsthe composition may be consumed more than once a day (e.g. once in themorning and once in the evening). The person skilled in the art willappreciate that the frequency of consumption can be adjusted to takeaccount of the concentration of macular pigment carotenoids, especiallymeso-zeaxantion, present in the formulation. The method of the inventioncan be adjusted accordingly.

Consuming the composition of the invention, or performing the method ofinvention, over a sufficient period of time (typically at least 8 weeks,preferably at least 3 months, more preferably over at least 6 months,and most preferably for 12 months or more) may typically result in anincrease in the level of macular pigment in a subject.

The amount of increase in the level of macular pigment carotenoids inthe subject which is achieved by consumption of the composition maydepend on, for example, the level of macular pigment carotenoids presentin the subject's eyes prior to commencement of consumption of thecomposition. As described above, the inventors have found that there isa proportion of the population (about 10% or so) in Ireland which haveabnormally low levels of macular pigment and an abnormal distribution ofcarotenoid pigments within the macula, and it is anticipated thatsimilar subjects exist in other populations. Such people might beexpected to exhibit a substantial increase in the level of macularpigment following long term (i.e. 6 months or more) consumption of thecomposition of the invention.

Significantly, however, and surprisingly, the inventors have also foundthat at least some parameters of visual performance (e.g. lettercontrast sensitivity; glare disability) can be improved by consumptionof the composition of the invention without necessarily a correspondingincrease in the level of macular pigment.

In particular, the composition/method of the invention can produce adetectable improvement in the visual performance of a subject inconditions other than low light. For example, the composition/method ofthe invention can produce an improvement in the visual performance of asubject in conditions of illumination greater than 1 Cdm⁻²; moreespecially in photopic conditions (e.g. illumination levels greater thanor equal to 3 Cdm⁻²).

More especially, the composition/method of the invention can produce animprovement in one or more of the following visual performanceparameters: visual acuity, especially best corrected visual acuity(BCVA); contrast sensitivity (CS); and glare disability (GD). Suitablemethods of measuring these visual performance parameters are known tothose skilled in the art and are described in detail herein. Typicallythe method/composition of the invention will produce an improvement ofat least 5%, preferably at least 8%, more preferably at least 10%,relative to the same parameter measured prior to consumption of thecomposition/performance of the method of the invention.

For the avoidance of doubt it is hereby explicitly stated that anyfeature of the invention described herein as preferable, advantageous,convenient, desirable, typical or the like may be present in anyembodiments of the invention in isolation, or in any combination withany one or more other such features, unless the context dictatesotherwise. In addition, features described in relation to one aspect ofthe invention will equally apply to the other aspects of the invention,unless the context dictates otherwise.

The content of all publications and citations mentioned in thisspecification is specifically incorporated herein by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described by way of illustrativeembodiment and with reference to the accompanying drawings, in which:

FIG. 1 is a schematic representation of the structural formulae forlutein, zeaxanthin and MZ;

FIG. 2 is a graph of corrected visual acuity against central macularpigment OD (arbitrary units) in a group of mixed normal and AMDsubjects;

FIG. 3 is a graph of macular pigment OD (at 0.25° eccentricity) againsttime for subjects consuming various macular carotenoid compositions;

FIG. 4 is a graph showing the macular pigment OD measurement, at varyingdegrees of eccentricity, for particular subjects found to have atypicalMPOD profiles, with a “central dip” (i.e. lower levels of macularpigment in the centre of the macula); mean±SD MPOD values showing the‘central dip’ in the spatial profile of MP. Temporal MPOD values weremeasured at 0.25°, 0.5°, 1°, 1.75°, 3° of eccentricity. Specifically,given the known symmetry of MPOD, the MPOD values for negativeeccentricities were assumed to be the same and constructed for thepurpose of illustration;

FIG. 5 is a graph of MPOD against time, for subjects receiving one ofthree different macular carotenoid formulations, wherein mean macularpigment optical density is measured at 0.25° retinal eccentricity atbaseline, 4 weeks, and 8 weeks according to group wise; n=31; Group 1:high L group; Group 2: mixed carotenoid group; Group 3: high MZ group;

FIG. 6 is a graph of MPOD against time, for subjects receiving one ofthree different macular carotenoid formulations, wherein mean macularpigment optical density is measured at 0.50° retinal eccentricity atbaseline, 4 weeks, and 8 weeks according to group wise; n=31; Group 1:high L group; Group 2: mixed carotenoid group; Group 3: high MZ group;

FIG. 7 is a graph of mean MPOD against retinal eccentricity for Group 1respectively (see example 2), before and after an 8 week period ofdietary supplementation, wherein the mean macular pigment opticaldensity spatial profile of Group 1 is measured at baseline (presupplementation) and at 8 weeks (post supplementation); mean+standarddeviation; n=11; Group 1: high L group;

FIG. 8 is a graph of mean MPOD against retinal eccentricity for Group 2(see example 2), before and after an 8 week period of dietarysupplementation, wherein the mean macular pigment optical densityspatial profile of Group 2 is measured at baseline (pre supplementation)and at 8 weeks (post supplementation); mean+standard deviation; n=10;Group 2: combined carotenoid group;

FIG. 9 is a graph of mean MPOD against retinal eccentricity for Group 3(see example 2), before and after an 8 week period of dietarysupplementation, wherein the mean macular pigment optical densityspatial profile of Group 3 is measured at baseline (pre supplementation)and at 8 weeks (post supplementation); mean+standard deviation; n=10;Group 3: high MZ group;

FIG. 10 is a graph of MPOD against retinal eccentricity (mean of eightsubjects; see example 5) before (circular symbols) or after (squaresymbols) 3 months of supplementation with a daily dose of 10 mg L, 10 mgMZ and 10 mg Z.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1 Comparison ofMacular Responses After Supplementation with Three Different MacularCarotenoid Formulations

Subjects and Recruitment

This study was conducted at the Institute of Vision Research, WhitfieldClinic, Waterford, Republic of Ireland. Seventy one subjects volunteeredto participate in this study, which was approved by the local researchethics committee. Subjects were aged between 32 to 84 years and in goodgeneral health. The volunteers were divided into two groups: an AMDgroup and a normal group. 34 subjects had confirmed early stage AMD inat least one eye (AMD group; categorized and identified by eitherpresence of drusen and/or pigmentary changes at the macula), and 37subjects had no ocular pathology (normal group). Importantly, for theAMD group, significant efforts were made to identify patients with earlyAMD who were not currently taking carotenoid supplements.

Study Design and Formulation

L=Lutein MZ=mesozeaxanthin Z=Zeaxanthin

This study was a single blind, randomized-controlled clinical trial oforal supplementation with three different macular carotenoidformulations, as follows:

Group 1: high L group

-   -   (n=24 [normal group=12 and AMD group=12];    -   L=20 mg/day, Z=2 mg/day);

Group 2: mixed carotenoid group

-   -   (n=24 [normal group=13 and AMD group=11];    -   MZ=10 mg/day, L=10 mg/day, Z=2 mg/day);

Group 3: high MZ group

-   -   (n=23 [normal group=12 and AMD group=11];    -   MZ=18 mg/day, L=2 mg/day L).    -   All subjects were instructed to take one capsule (oil based) per        day with a meal for 8 weeks. Compliance was assessed by tablet        counting at each study visit.

Measurement of Macular Pigment Optical Density (MPOD)

The spatial profile of MP was measured using customized heterochromaticflicker photometry (cHFP) using the Macular Densitometer™, a cHFPinstrument that is slightly modified from a device described by Wooten &Hammond (2005 Optometry & Vision Science 82, 378-386) and by Kirby etal., (2010 Invest. Ophthalmol. Vis. Sci. 51, 6722-6728.

Subjects were assessed at baseline, two weeks, four weeks, six weeks,and 8 weeks (V1, V2, V3, V4, and V5, respectively). MPOD was measured atthe following eccentricities: at 0.25°, 0.5°, 1°, 1.75°, 3° but onlyresults at 0.25°, the central part of the retina corresponding to themacula, are reported here.

Statistical Analysis The statistical software packages PASW Statistics17.0 (SPSS Inc., Chicago, Ill., USA) and R were used for analysis andSigma Plot 8.0 (Systat Software Inc., Chicago, Ill., USA) was used forgraphical presentations. All quantitative variables investigatedexhibited a typical normal distribution. We used the 5% level ofsignificance.

Results

MPOD and Visual Acuity

There was a positive and statistically significant relationship betweencentral MPOD (at 0.25°) and corrected visual acuity at baseline(r=0.303, p=0.008), as shown in FIG. 2 which is a graph of correctedvisual acuity against MPOD (arbitrary units), showing the data pointsfor individual subjects in the two groups prior to supplementation withone of the three carotenoid formulations. This finding suggests thatcentral MP is significantly and positively related to visualperformance.

Increase in MPOD Over Time

At an eccentricity of 0.25° the baseline MPOD was different for eachgroup as follows; Group 1: 0.42±0.20 Group 2: 0.44±0.18 Group3:0.49±0.21, with a mean of all groups of 0.45±0.20. To simplify thecomparison all groups are drawn to start at the mean value. The studyshowed an increase in MPOD over time, as illustrated in FIG. 3, which isa graph of MPOD at 0.25° eccentricity (arbitrary units) against time(timepoints 1 to 5, corresponding to 0, 2, 4, 6 & 8 weeks respectively).As seen in FIG. 3, the biggest increase in central MPOD was achievedwith the group 2 formulation (MZ=10 mg/day, L=10 mg/day, Z=2 mg/day)which was statistically significant different from groups 1 and 3. Therewas no statistical difference between group 1 and 3.

Conclusions

Surprisingly, the greatest effect on macular pigment was seen with themixed carotenoid group (group 2) containing MZ10 mg L10 mg Z 2 mg,whereas results with the other two groups were very similar. Thereappears to be synergism between MZ & L. That the high MZ group (group 3)was able to increase MP demonstrates that MZ can raise MPODsubstantially without any contribution from the other carotenoids, butwas less effective than MZ in combination with L.

Macular Carotenoid Supplementation in Subjects with ‘Central Dips’ inTheir Macular Pigment Spatial Profiles

The central retina, known as the macula, is responsible for color andfine-detail vision (Hirsh & Curcio 1989; Vision Res. 29, 1095-1101). Apigment of the two dietary carotenoids, lutein (L) and zeaxanthin (Z),and a typically non-dietary carotenoid MZ (MZ), (Johnson et al., 2005Invest. Ophtalmol. Vis. Sci. 46, 692-702) accumulates at the macula,where it is known as macular pigment (MP). MP is a blue light filter(Snodderly et al., 1984 Invest. Opthalmol. Vis. Sci. 25, 660-673) and apowerful antioxidant (Khachik et al. 1997 Invest. Ophthalm. & Vis. Sci.38, 1802-1811), and is therefore believed to protect against age-relatedmacular degeneration (AMD), which is now the most common cause of blindregistration in the western world (Klaver et al., 1998 Arch. Ophthalmol.116, 653-658).

MZ and Z are the predominant carotenoids in the foveal region, whereas Lpredominates in the parafoveal region (Snodderley et al., 1991 Invest.Ophthalmol. Vis. Sci. 32, 268-279). The concentration of MZ peakscentrally, with an MZ:Z ratio of 0.82 in the central retina (within 3 mmof the fovea) and 0.25 in the peripheral retina (11-21 mm from thefovea) (Bone et al., 1997 Experimental Eye Research 64, 211-218).Retinal MZ is produced primarily by isomerization of retinal L, thusaccounting for lower relative levels of L, and higher relative levels ofMZ, in the central macula, and vice versa in the peripheral macula, andwould also explain why MZ accounts for about one third of total MP.

The concentration of MP varies greatly amongst individuals (Hammond etal., 1997 Journal of the Optical Society of America A-Optics ImageScience & Vision, 14, 1187-1196). Atypical MP spatial profiles (i.e.‘central dips’) are present in some individual MP profiles. Moreimportantly, it was confirmed that these ‘central dips’ were real andreproducible features of the MP spatial profile, when measured usingcustomised heterochromatic flicker photometry (cHFP, a validatedtechnique for measuring MP). The importance of such variations, if any,in the spatial profile of MP (e.g. the presence of a ‘central dip’) isnot yet known, but may be related to the putative protective role ofthis pigment. For example, reduced MPOD at the centre of the macula(i.e. the presence of a ‘central dip’) may be associated with increasedrisk of developing AMD.

It has been shown that 12% (58 subjects out of a sample database of 484subjects) of the normal Irish population had a reproducible ‘centraldip’ in their MPOD spatial profile and that such a dip in the MP spatialprofile is more common in older subjects and in cigarette smokers (twoof the established risk factors for AMD).

Example 2 Supplementation of Formulations Containing Macular Carotenoidsto Subjects with a “Central Dip” in Their MP Profile

The study described in this example was performed with volunteersubjects from the above mentioned database (n=58) in the “central dipstudy”, who were identified, and confirmed, as having ‘central dips’ intheir MP spatial profile (i.e. MPOD at 0.5 degrees of eccentricity was≧MPOD at 0.25 degrees of eccentricity, see FIG. 4) and invited toparticipate in an 8-week supplementation trial with one of threedifferent macular carotenoid formulations (see below).

Methods

Subjects and Study Design:

Fifty eight subjects with ‘central dips’ in their MP spatial profile(identified from a master MP database; n=484) were invited to take partin the study. Of the 40 subjects that agreed to come back for testing,31 were confirmed as still having a ‘central dip’ (i.e. MPOD at 0.5degrees of eccentricity was ≧MPOD at 0.25 degrees of eccentricity) andwere therefore enrolled into the 8-week supplementation trial.

All subjects signed an informed consent document and the experimentalmeasures conformed to the Declaration of Helsinki. The study wasreviewed and approved by the Research Ethics Committee, WaterfordInstitute of Technology, Waterford, Ireland. Inclusion criteria forparticipation in this study were as follows: MPOD at 0.5 degrees ofeccentricity ≧MPOD at 0.25 degrees of eccentricity (i.e. evidence of a‘central dip’ in the MP spatial profile); no presence of ocularpathology; visual acuity 20/60 or better in the study eye; not currentlytaking L and/or Z and/or MZ dietary supplements.

Subjects were randomly assigned into one of the three groups as follows;

Group 1: high L group (n=11), L=20 mg/day, Z=2 mg/day;

Group 2: mixed carotenoid group (n=10), MZ=10 mg/day, L=10 mg/day, Z=2mg/day.

Group 3 the high MZ group (n=10), 18 mg/day MZ, 2 mg/day L).

All subjects were instructed to take one capsule per day with a meal for8 weeks. MPOD, including its spatial profile, i.e. at 0.25°, 0.5°, 1°,1.75°, 3°, was measured at baseline, four weeks and 8 weeks.

Measurement of Macular Pigment Optical Density

The spatial profile of MP was measured using cHFP using the MacularDensitometer™, as described in Example 1. In order to measure thespatial profile of MP, measurements were made at the following degreesof retinal eccentricity: 0.25°, 0.5°, 1°, 1.75°, 3° and 7° (thereference point) obtained using the following sized target diameters; 30minutes, 1°, 2°, 3.5°, 1° and 2°,

Statistical Analysis

The statistical software package PASW Statistics 17.0 (SPSS Inc.,Chicago, Ill., USA) was used for analysis and Sigma Plot 8.0 (SystatSoftware Inc., Chicago, Ill., USA) was used for graphical presentations.All quantitative variables investigated exhibited a typical normaldistribution. Means±SDs are presented in the text and tables.Statistical comparisons of the three different intervention groups, atbaseline, were conducted using independent samples t-tests andchi-square analysis, as appropriate. We used the 5% level ofsignificance throughout our analysis.

Results

Change in MPOD Over 8-Week Supplementation Period

We conducted repeated measures ANOVA of MPOD, for all retinaleccentricities measured (i.e. at 0.25°, 0.5°, 1.0°, 1.75°, and 3°), overtime (i.e. over the study period [baseline, 4 weeks and 8 weeks]), usinga general linear model approach, with one between-subjects factor:treatment (Group 1, Group 2, Group 3) and age as a covariate. FIGS. 5and 6 show the change in MPOD during the course of the trial formeasurements at 0.25 and 0.5° eccentricity respectively. Table 1presents repeated measures ANOVA results for each group separately andfor each degree of retinal eccentricity. As seen in this Table, increasein MPOD at 0.25° and 0.5° was statistically significant in Group 2 (i.e.the mixed carotenoids group). Similarly, a significant increase in MPODat 0.25° was seen in Group 3 (i.e. high MZ group). Of note, only theincrease in MPOD at 0.25° in Group 2 remains significant afterBonferroni correction for multiple testing.

Change in the spatial profile of MPOD for each of Groups 1-3 isillustrated in FIGS. 7-9 respectively.

Conclusions

Only the two formulations containing MZ were able to correct the“central dip” and increase MP. Surprisingly, and contrary toexpectation, the formulation containing L but without MZ had no effecton MPOD at any eccentricity.

The formulation containing mixed carotenoids (group 2) had a superioreffect since it increased MP significantly at both 0.25 and 0.5eccentricities. This is consistent with the result from the subjects whoreceived a supplement with all three carotenoids without a central dipat the baseline (see Example 1) i.e. the greatest response was observedusing a supplement containing each of MZ, L and Z.

TABLE 1 Average MPOD values at each degree of eccentricity for allsubjects according to group & visit wise Time interaction Group MPODBaseline 4 wks 8 wks (p-value) Group 1 0.25 0.45 ± 0.20 0.48 ± 0.22 0.49± 0.21 0.112 Group 1 0.5 0.45 ± 0.23 0.46 ± 0.18 0.46 ± 0.23 0.509 Group1 1 0.20 ± 0.18 0.27 ± 0.15 0.25 ± 0.13 0.234 Group 1 1.75 0.15 ± 0.090.15 ± 0.09 0.15 ± 0.09 0.986 Group 1 3 0.15 ± 0.11 0.16 ± 0.09 0.11 ±0.08 0.265 Group 2 0.25 0.41 ± 0.27 0.50 ± 0.27 0.59 ± 0.30 0.000 Group2 0.5 0.44 ± 0.26 0.46 ± 0.28 0.52 ± 0.28 0.016 Group 2 1 0.26 ± 0.230.29 ± 0.15 0.34 ± 0.10 0.417 Group 2 1.75 0.18 ± 0.10 0.19 ± 0.06 0.22± 0.06 0.218 Group 2 3 0.16 ± 0.12 0.14 ± 0.06 0.19 ± 0.11 0.448 Group 30.25 0.48 ± 0.16 0.55 ± 0.19 0.57 ± 0.18 0.005 Group 3 0.5 0.48 ± 0.150.48 ± 0.17 0.50 ± 0.15 0.786 Group 3 1 0.32 ± 0.12 0.31 ± 0.13 0.34 ±0.12 0.596 Group 3 1.75 0.11 ± 0.09 0.12 ± 0.07 0.13 ± 0.08 0.743 Group3 3 0.12 ± 0.08 0.15 ± 0.07 0.15 ± 0.07 0.522

Values represent mean±standard deviation; n=31; MPOD=macular pigmentoptical density; 0.25°=MPOD measured at 0.25° retinal eccentricity;0.5°=MPOD measured at 0.5° retinal eccentricity; 1.0°=MPOD measured at1.0° retinal eccentricity; 1.75°=MPOD measured at 1.75° retinaleccentricity; 3°=MPOD measured at 3.0° retinal eccentricity; Group 1:high L group; Group 2: combined carotenoid group; Group 3: high MZgroup; the p-values represent repeated measures ANOVA for the 3 studyvisits (within-subject effects), with Greenhouse-Gesser correction forlack of sphericity as appropriate.

Example 3 Comparison of Visual Performance in Subjects with Early StageAMD After Supplementation with Three Different Macular CarotenoidFormulations

Subjects and Recruitment

This study was conducted with 72 subjects, many with early AMD. Fordetails see Example 1.

Study Design and Formulation

The subjects with were divided into 3 groups (of 20-27 subjects) andgiven the following supplementations:

Group 1: L=20; Z=2 mg/day

Group 2: L=10; MZ=10; Z=2 mg/day

Group 3: MZ=17-18; L=2-3; Z=2 mg/day

These formulations, dissolved in 0.3 ml vegetable oil, were administeredin soft gel capsules.

Visual Performance, using the techniques described previously above, wasmeasured at baseline and at 3 and 6 months after supplementation.Statistical analyses were performed using a paired t test. Significantvalues were considered as P<0.05. Results are only given where at leastone group was statistically significant.

Results:

Since there were no statistically significant improvements detected inVP after 3 months treatment, only results for 6 or 12 months arepresented here (below):

1. Baseline Comparison Between Groups:

TABLE 2 Baseline Comparison Group 2: Group 1: 10 mg MZ; Group 3: 20 mgL; 10 mg L; 18 mg MZ; Variable 2 mg Z 2 mg Z 2 mg L p N 23 27 22 Age 67± 8 64 ± 9  72 ± 10 0.014 MPOD 0.25° 0.412 ± 0.19 0.482 ± 0.21 0.475 ±0.20 0.411 BCVA  92 ± 21  97 ± 10 94 ± 8 0.362

The groups were statistically comparable at baseline with respect to MPand vision (assessed by Best Corrected Visual Acuity, “BCVA”). There wasa significant difference between groups at baseline for age betweenGroup 3 and the other two Groups. Group 1 and Group 2 were statisticallysimilar with respect to age.

2. Best Corrected Visual Acuity (BCVA)

There was a baseline correlation (before supplementation) of a positiveand statistically significant relationship between central MPOD (0.25)and BCVA, importantly this is in the AMD population (r−=0.368, p=0.002).There was no statistically significant change in BCVA in any group after3 and 6 months.

A computer-generated LofMAR test chart (Test Chart 2000 Pro; ThomsonSoftware Solutions) was used to determine BCVA at a viewing distance of4 m, using a Sloan ETDRS letterset. BCVA was determined as the averageof three measurements, with letter and line changes facilitated by thesoftware pseudo-randomization feature. Best corrected visual acuity wasrecorded using a letter-scoring visual acuity rating, with 20/20 (6/6)visual acuity assigned a value of 100. Best corrected visual acuity wasscored relative to this value, with each letter correctly identifiedassigned a nominal value of one, so that, for example, a BCVA of 20/20+1(6/6+1) equated to a score of 101, and 20/20−1 (6/6−1) to 99.

3. MPOD Response

Table 3 below presents MP data for each Group and for each eccentricitymeasured, at baseline, six and twelve months after supplementation withmacular carotenoids.

A statistically significant increase in MPOD at 12 months was observedonly in groups 2 and 3, receiving the MZ-containing supplement.

TABLE 3 MPOD MPOD MPOD Group Baseline 6 months 12 months 0.25 0.25 p 1:0.42 ± 0.19 0.51 ± 0.20 0.57 ± 0.30 0.148 2: 0.48 ± 0.22 0.58 ± 0.210.63 ± 0.19 0.001 3: 0.52 ± 0.20 0.58 ± 0.22 0.57 ± 0.20 0.022 0.5  0.5 p 1: 0.32 ± 0.19 0.42 ± 0.18 0.46 ± 0.27 0.126 2: 0.39 ± 0.19 0.50 ±0.18 0.52 ± 0.19 0.001 3: 0.41 ± 0.19 0.46 ± 0.19 0.45 ± 0.20 0.034 1.0 1.0  p 1: 0.22 ± 0.11 0.31 ± 0.15 0.32 ± 0.17 0.213 2: 0.25 ± 0.12 0.36± 0.17 0.37 ± 0.18 0.001 3: 0.26 ± 0.15 0.32 ± 0.14 0.33 ± 0.16 0.0251.75 1.75 p 1: 0.13 ± 0.10 0.18 ± 0.11 0.20 ± 0.10 0.114 2: 0.14 ± 0.100.22 ± 0.12 0.24 ± 0.11 <0.001   3: 0.13 ± 0.11 0.21 ± 0.12 0.19 ± 0.100.063

4. Letter Contrast Sensitivity (Thomson Chart)

Table 4A presents letter contrast sensitivity data at baseline and sixmonths after supplementation with macular carotenoids Measurements weremade at 1.2, 2.4, 6.0, and 9.6 cpd. There was a statisticallysignificant improvement only in Group 2 (10 mg MZ; 10 mg L; 2 mg Z) at1.2, 2.4 and 9.6 cpd and not at all in the other two groups. This showsa greatly superior effect in Group 2.

TABLE 4A Letter contrast Letter contrast sensitivity sensitivity GroupBaseline Six months 1.2 cpd 1.2 cpd P 1: 1.68 ± 0.34 1.75 ± 0.30 0.0912: 1.63 ± 0.24 1.80 ± 0.25 0.013 3: 1.68 ± 0.37 1.63 ± 0.25 0.322 2.4cpd 2.4 cpd p 1: 1.60 ± 0.33 1.66 ± 0.34 0.17  2: 1.59 ± 0.29 1.72 ±0.33 0.049 3: 1.61 ± 0.35 1.63 ± 0.32 0.6  9.6 cpd 9.6 cpd p 1:  1.1 ±0.36 1.04 ± 0.41 0.194 2: 0.97 ± 0.32 1.11 ± 0.46 0.043 3: 0.94 ± 0.370.95 ± 0.43 0.901

Table 4B shows the letter contrast sensitivity (CS) at baseline and 12months, for each of five spatial frequencies (1.2-15.15 cpd).

TABLE 4B Mean (±sd) letter contrast sensitivity (CS) values at baselineand at 12 months. Group 1 Group 2 Group 3 cpd Baseline 12 months pBaseline 12 months p Baseline 12 months P 1.2 1.74 ± 0.31 1.86 ± 0.300.033 1.69 ± 0.24 1.88 ± 0.28 0.004 1.73 ± 0.30 1.89 ± 0.27 0.041 2.41.65 ± 0.32 1.79 ± 0.38 0.013 1.66 ± 0.28 1.79 ± 0.31 0.004 1.60 ± 0.301.85 ± 0.29 0.002 6.0 1.37 ± 0.29 1.42 ± 0.40 0.194 1.30 ± 0.29 1.38 ±0.33 0.053 1.19 ± 0.43 1.55 ± 0.27 0.002 9.6 1.11 ± 0.28 1.09 ± 0.340.775 1.00 ± 0.32 1.10 ± 0.40 0.034 0.91 ± 0.45 1.19 ± 0.40 0.012 15.150.73 ± 0.33 0.73 ± 0.39 0.933 0.64 ± 0.37 0.73 ± 0.49 0.148 0.57 ± 0.460.83 ± 0.36 0.014 Abbreviations: cpd = cycles per degree

At 12 months the results were similar to 6 months in that lettercontrast sensitivity increased in all groups for large objects (1.2 and2.4 cpd) but only in groups 2 and 3 with smaller objects (6.0-15.5 cpd).

Table 4C reports the relationship between observed changes in MPOD (at0.25° eccentricity) and observed changes in letter CS at 1.2 cpd. Ofnote, there were no statistically significant relationships betweenchange in MP and change in letter CS, at any spatial frequency.

TABLE 4C Change in MPOD vs. change in letter CS r p Group 1 0.262 0.294Group 2 0.258 0.235 Group 3 −0.043 0.875

Colour Fundus Photographs

Colour fundus photographs were taken at every study visit using a ZeissVisuCam™ (Carl Zeiss Meditec A G, Jena, Germany) and were gradedstereoscopically at the Ocular Epidemiology Reading Center at theUniversity of Wisconsin, USA. Photographs were graded using a modifiedversion of the Wisconsin Age-Related Maculopathy Grading System. EarlyAMD was defined as the presence of drusen and/or pigmentary changes inat least one eye, confirmed by an on-site ophthalmologist incollaboration with graders at the University of Wisconsin. Each fundusphotograph was evaluated, lesion-by-lesion, to determine maximum drusensize, type, area, and retinal pigmentary abnormalities. Overall findingswere reported on an 11-step AMD severity scale. A change of two or moresteps along the severity scale was defined as being clinicallysignificant. Graded photographs were obtained for baseline and 12 monthsvisits.

At baseline, there was no significant difference between the groups withrespect to AMD grade (p=0.679) [Table 4D].

TABLE 4D AMD grading for entire groups and subgroups at baseline. Entiregroup Group 1 Group 2 Group 3 Grade (n = 72) (n = 23) (n = 27) (n = 22)Sig. 1-3 16 (22.2%) 7 (30.4%) 6 (22.2%) 3 (13.6%) 0.679 4-5 28 (38.9%)10 (43.5%) 8 (29.6%) 10 (45.5%) 6-7 19 (26.4%) 5 (21.7%) 8 (29.6%) 6(27.3%) 8-9 4 (5.6%) — 2 (7.4%) 2 (9.1%) 10-11 5 (6.9%) 1 (4.3%) 3(11.1%) 1 (4.5%)

The changes in AMD grade between baseline and 12 months for each of thethree groups are summarized in Table 4E. A change in the negativedirection (i.e. −1, −2) indicates a progression along the AMD severityscale, whereas positive integers indicate regression (improvement) alongthe AMD severity scale. Between baseline and 12 months, there was nostatistically significant difference between treatment groups withrespect to change in AMD severity (p=0.223, Pearson chi-square test).

TABLE 4E Change in AMD grade (11-step scale) between baseline and 12months. Group n −2 −1 0 +1 +2 Sig. 1 16 1 (6%) 1 (6%)  10 3 (19%) 10.223 (63%) (6%) 2 23 1 (4%) 2 (9%)  14 4 (17%) 2 (61%) (9%) 3 15  2(13%) 6 (40%)  4 2 (13%) 1 (27%) (7%) Total 54 4 (7%) 9 (17%) 28 9 (17%)4 (100%) (52%) (7%) Abbreviations: n = number of subjects; negativevalue indicates disease progression; a positive value indicates diseaseregression; 0 = no change in grade

Of note, table 4E shows that 86% of subjects exhibited no clinicallysignificant change in the status of their AMD between baseline and 12months, with 7% exhibiting deterioration and 7% exhibiting animprovement (note: a change in grade of two or more has been accepted asbeing clinically significant).

Discussion

The most interesting results were for letter contrast sensitivity. Thistest is only conducted in daylight and tests letters of different sizes.Results were at 6 months and 12 months were similar. There was nocorrelation between increase in MP and increase in this parameterindicating a neuro-physiological effects of macular carotenoids.

There was no significant change in AMD grade from baseline. Thus changesin contrast sensitivity were not related to effects on AMD pathology.

5. Contrast Sensitivity at Night (Assessed on the FACT Device)

Table 5 below presents log contrast sensitivity data assessed for nighttime, at baseline and six months after supplementation with macularcarotenoids. Measurements were made at 1.5, 3.0, 6.0, 12 and 18 cpm. Thestatistically significant improvement in this measure of VP was presentonly in Group 2 at 1.5, 3.0, cpd and in Group 1 at 1.5 cpd showing asuperior effect of group 2.

TABLE 5 Night time contrast Night time contrast sensitivity sensitivityGroup Baseline Six months 1.5 cpd 1.5 cpd P 1: 1.53 ± 0.29 1.67 ± 0.260.124 2: 1.51 ± 0.27 1.66 ± 0.3  0.028 3: 1.44 ± 0.29 1.45 ± 0.34 0.9113.0 cpd 3.0 cpd p 1: 1.52 ± 0.25  1.8 ± 0.28 0.001 2: 1.62 ± 0.34 1.75 ±0.41 0.01  3: 1.55 ± 0.40  1.6 ± 0.41 0.585

6. Contrast Sensitivity at Daytime (Assessed on the FACT Device)

Table 6 below presents log contrast sensitivity data assessed for daytime at baseline and six months after supplementation with macularcarotenoids. Measurements were made at 1.5, 3.0. 6.0, 12 and 18 cpm. Thestatistically significant improvement in this measure of VP was presentin Group 2 at 1.5, 3.0, and 18 cpd and in Group 1 at 1.5 cpd showing asuperior effect in group 2.

TABLE 6 Daytime contrast Daytime contrast sensitivity sensitivity GroupBaseline Six months 1.5 cpd 1.5 cpd P 1: 1.41 ± 0.16 1.57 ± 0.26 0.03 2: 1.48 ± 0.23  1.6 ± 0.28 0.034 3: 1.41 ± 0.13  1.5 ± 0.28 0.238 3.0cpd 3.0 cpd p 1: 1.67 ± 0.21 1.75 ± 0.21 0.17  2:  1.7 ± 0.33 1.81 ±0.34 0.018 3: 1.72 ± 0.18 1.77 ± 0.29 0.46   18 cpd  18 cpd p 1: 0.62 ±0.4  0.56 ± 0.41 0.497 2: 0.65 ± 0.38 0.77 ± 0.5  0.015 3: 0.57 ± 0.4 0.62 ± 0.43 0.704

7. Contrast Sensitivity at Night Time in the Presence of Glare (Assessedon the FACT Device)

Table 7 below presents Log contrast sensitivity data at night in thepresence of glare at baseline and six months after supplementation withmacular carotenoids. Measurements were made at 1.5, 3.0, 6.0, 12 and 18cpd. There was a statistically significant improvement in this VP onlyin Group 2 at 18 cpd.

TABLE 7 Night time contrast Night time contrast sensitivity sensitivitywith glare with glare Baseline Six months Group 18 cpd 18 cpd p 1: 0.34± 0.16 0.34 ± 0.16 0.136 2: 0.36 ± 0.13 0.47 ± 0.34 0.038 3: 0.36 ± 0.220.32 ± 0.08 0.588

8. Contrast Sensitivity at Day Time in the Presence of Glare (Assessedon the FACT Device)

Table 8 below presents Log contrast sensitivity data at day time in thepresence of glare, at baseline and six months after supplementation withmacular carotenoids. Measurements were made at 1.5, 3.0, 6.0, 12 and 18cpd. The statistically significant improvement in this measure of VP waspresent in Group 2 at 1.5, 3.0, 6.0, and 18 cpd cpd and in Group 1 at1.5, 3.0, and 6.0 cpd and in Group 3 at 6 cpd, showing a superior effectin group 2.

TABLE 8 Daytime contrast Daytime contrast sensitivity with sensitivitywith glare glare Group Baseline Six months 1.5 cpd 1.5 cpd P 1:  1.5 ±0.25 1.63 ± 0.21 0.001 2: 1.43 ± 0.25 1.68 ± 0.24 0.002 3: 1.42 ± 0.381.46 ± 0.36 0.351 3.0 cpd 3.0 cpd p 1: 1.68 ± 0.22 1.85 ± 0.22 0.006 2:1.71 ± 0.25 1.84 ± 0.25 0.007 3: 1.67 ± 0.35 1.71 ± 0.43 0.542 6.0 cpd6.0 cpd p 1: 1.46 ± 0.42 1.85 ± 0.22 <0.001   2: 1.46 ± 0.47 1.84 ± 0.25<0.001   3: 1.36 ± 0.42 1.71 ± 0.43 0.001  18 cpd  18 cpd p 1: 0.64 ±0.46 0.59 ± 0.43 0.642 2: 0.53 ± 0.32 0.67 ± 0.51 0.018 3:  0.7 ± 0.470.66 ± 0.45 0.609

9. Contrast Sensitivity and Glare Disability Between Baseline and 12Months

Data on contrast sensitivity (CS) and glare disability (GD) undermesopic (night-time) and photopic (daytime) conditions, at baseline and12 months, are presented in Tables 9-12.

TABLE 9 Log CS at baseline and 12 months under mesopic conditions (FACTdevice) Group p CS 1.5 cpd v1 CS 1.5 cpd v4 Group 1 1.59 ± 0.28 1.80 ±0.22 0.007 Group 2 1.60 ± 0.27 1.76 ± 0.24 0.047 Group 3 1.53 ± 0.391.73 ± 0.25 0.124 CS 3 cpd v1 CS 3 cpd v4 Group 1 1.61 ± 0.25 1.82 ±0.22 0.007 Group 2 1.68 ± 0.34 1.80 ± 0.26 0.058 Group 3 1.62 ± 0.421.85 ± 0.40 0.175 CS 6 cpd v1 CS 6 cpd v4 Group 1 1.18 ± 0.38 1.24 ±0.53 0.521 Group 2 1.27 ± 0.40 1.38 ± 0.44 0.278 Group 3 1.20 ± 0.441.46 ± 0.50 0.060 CS 12 cpd v1 CS 12 cpd v4 Group 1 0.65 ± 0.14 0.79 ±0.43 0.224 Group 2 0.67 ± 0.26 0.79 ± 0.24 0.080 Group 3 0.76 ± 0.250.89 ± 0.36 0.177 CS 18 cpd v1 CS 18 cpd v4 Group 1 0.40 ± 0.25 0.32 ±0.08 0.207 Group 2 0.32 ± 0.07 0.36 ± 0.26 0.332 Group 3 0.36 ± 0.150.39 ± 0.24 0.476 Abbreviations: FACT = functional acuity contrast test;CS = contrast sensitivity; cpd = cycles per degree; v1 = baseline visit;v4 = 12 month visit

TABLE 10 Log CS at baseline and 12 months under photopic conditions(FACT device) Group p CS 1.5 cpd v1 CS 1.5 cpd v4 Group 1 1.47 ± 0.251.63 ± 0.22 0.007 Group 2 1.56 ± 0.21 1.61 ± 0.24 0.478 Group 3 1.44 ±0.22 1.63 ± 0.25 0.023 CS 3 cpd v1 CS 3 cpd v4 Group 1 1.70 ± 0.22 1.86± 0.11 0.002 Group 2 1.74 ± 0.33 1.86 ± 0.21 0.108 Group 3 1.78 ± 0.201.84 ± 0.24 0.402 CS 6 cpd v1 CS 6 cpd v4 Group 1 1.52 ± 0.30 1.59 ±0.29 0.310 Group 2 1.52 ± 0.39 1.66 ± 0.39 0.064 Group 3 1.44 ± 0.451.62 ± 0.38 0.192 CS 12 cpd v1 CS 12 cpd v4 Group 1 1.01 ± 0.33 0.98 ±0.35 0.709 Group 2 1.02 ± 0.36 1.21 ± 0.48 0.118 Group 3 0.99 ± 0.431.19 ± 0.48 0.164 CS 18 cpd v1 CS 18 cpd v4 Group 1 0.63 ± 0.39 0.54 ±0.40 0.437 Group 2 0.59 ± 0.38 0.64 ± 0.48 0.687 Group 3 0.68 ± 0.480.76 ± 0.50 0.458 Abbreviations: FACT = functional acuity contrast test;GD = glare disability; cpd = cycles per degree; v1 = baseline visit; v4= 12 month visit

TABLE 11 Log GD at baseline and 12 months under mesopic conditions (FACTdevice) Group p GD 1.5 cpd v1 GD 1.5 cpd v4 Group 1 1.49 ± 0.37 1.52 ±0.34 0.635 Group 2 1.44 ± 0.39 1.53 ± 0.35 0.365 Group 3 1.26 ± 0.441.53 ± 0.47 0.029 GD 3 cpd v1 GD 3 cpd v4 Group 1 1.57 ± 0.43 1.60 ±0.32 0.728 Group 2 1.51 ± 0.38 1.70 ± 0.35 0.010 Group 3 1.39 ± 0.501.55 ± 0.49 0.346 GD 6 cpd v1 GD 6 cpd v4 Group 1 1.09 ± 0.37 1.04 ±0.34 0.564 Group 2 1.18 ± 0.35 1.24 ± 0.43 0.581 Group 3 1.10 ± 0.401.20 ± 0.47 0.348 GD 12 cpd v1 GD 12 cpd v4 Group 1 0.66 ± 0.17 0.71 ±0.18 0.343 Group 2 0.66 ± 0.17 0.80 ± 0.43 0.100 Group 3 0.77 ± 0.240.69 ± 0.22 0.115 GD 18 cpd v1 GD 18 cpd v4 Group 1 0.34 ± 0.16 0.30 ±0.00 0.336 Group 2 0.34 ± 0.10 0.39 ± 0.37 0.483 Group 3 0.32 ± 0.080.36 ± 0.21 0.336 Abbreviations: FACT = functional acuity contrast test;GD = glare disability; cpd = cycles per degree; v1 = baseline visit; v4= 12 month visit

TABLE 12 Log GD at baseline and 12 months under photopic conditions(FACT device) Group p GD 1.5 cpd v1 GD 1.5 cpd v4 Group 1 1.60 ± 0.251.76 ± 0.23 0.006 Group 2 1.53 ± 0.19 1.74 ± 0.22 0.002 Group 3 1.51 ±0.25 1.69 ± 0.42 0.058 GD 3 cpd v1 GD 3 cpd v4 Group 1 1.70 ± 0.26 1.89± 0.25 0.002 Group 2 1.78 ± 0.21 1.97 ± 0.18 0.001 Group 3 1.73 ± 0.201.84 ± 0.38 0.330 GD 6 cpd v1 GD 6 cpd v4 Group 1 1.54 ± 0.38 1.64 ±0.35 0.358 Group 2 1.56 ± 0.43 1.69 ± 0.34 0.087 Group 3 1.46 ± 0.471.71 ± 0.38 0.048 GD 12 cpd v1 GD 12 cpd v4 Group 1 1.02 ± 0.42 1.05 ±0.38 0.659 Group 2 0.97 ± 0.36 1.14 ± 0.35 0.169 Group 3 1.00 ± 0.441.11 ± 0.43 0.320 GD 18 cpd v1 GD 18 cpd v4 Group 1 0.64 ± 0.45 0.67 ±0.48 0.752 Group 2 0.54 ± 0.34 0.81 ± 0.51 0.071 Group 3 0.75 ± 0.480.75 ± 0.52 0.993 Abbreviations: FACT = functional acuity contrast test;GD = glare disability; cpd = cycles per degree; v1 = baseline visit; v4= 12 month visit

Discussion

Results at 12 months were similar to those at 6 months, in that theresults were variable and difficult to interpret. Under mesopic(nighttime) conditions, contrast sensitivity only increased with largeobjects (1.5 and 3.0 cpd) in groups 1 and 2. For glare disability, group1 did not change, whilst group 2 and 3 showed some change with largeobjects.

Under photopic (daylight) conditions, groups 1 and 3 only increasedcontrast sensitivity with large objects. With glare disability allgroups increased only with large objects.

10. Changes in Visual Performance Parameters and Changes in MPOD

Table 13 reports the relationship between observed changes in MPOD (at0.25° eccentricity) and observed changes in parameters of visualperformance, namely CDVA and measures of mesopic and photopic contrastsensitivity, and mesopic and photopic glare disability, at 1.5 cpd. Ofnote, there were no statistically significant relationships betweenchange in MP and change in visual performance in any of the groups (withthe exception of a negative relationship between increases in MPOD andphotopic CS at 1.5 cpd in Group 1 only).

TABLE 13 r p Change in MPOD vs. change in CDVA Group 1 −0.320 0.211Group 2 −0.148 0.558 Group 3 −0.126 0.681 Change in MPOD vs. change inmesopic CS 1.5 cpd Group 1 0.055 0.859 Group 2 −0.140 0.664 Group 30.041 0.906 Change in MPOD vs. change in photopic CS 1.5 cpd Group 1−0.705 0.007 Group 2 −0.106 0.743 Group 3 −0.122 0.720 Change in MPODvs. change in mesopic GD 1.5 cpd Group 1 0.318 0.289 Group 2 −0.1060.743 Group 3 0.388 0.238 Change in MPOD vs. change in photopic GD 1.5cpd Group 1 −0.262 0.388 Group 2 −0.136 0.673 Group 3 −0.308 0.357Abbreviations: MPOD = macular pigment optical density; CDVA = correcteddistance visual acuity; L = lutein; Z = zeaxanthin; MZ =meso-zeaxanthin; CS = contrast sensitivity; cpd = cycles per degree; GD= glare disability.

Discussion

There was no correlation between increases in visual performance andincreases in macular pigment, indicating a neuro-physiological effect ofmacular carotenoids.

Other Conclusions: Changes in VP were Only Statistically SignificantAfter 6 Months or More

The methods reported here in contrast sensitivity were at varyingspatial frequencies. Low spatial frequencies (e.g. 1.2 cpd) areindicative of very large objects (e.g. a car, a house), whereas, largespatial frequencies (e.g. 18 cpd) are indicative of small objects (e.g.a menu in a restaurant). The data lead to the following conclusions;

1. The most important effect was on contrast sensitivity which is one ofthe most important measures of VP and it reflects how the patientactually perceives their own vision.

2. Statistical significance was reached across many spatial frequencies,which means the improvement detected has implications for general andreal life vision.

3. There was a superior improvement in VP for the Group 2 intervention(i.e. 10 mg MZ; 10 mg L; 2 mg Z).

Example 4 Effect of Two Macular Carotenoids and a Placebo Formulationson VP in Normal Subjects

Subjects and Recruitment

This study was conducted on 36 normal subjects with no AMD. Details ofthe recruitment are given in Example 1. Of the 36 subjects recruited, 32completed the trial, with one drop-out from each of the interventiongroups and two drop-outs from group 3, the placebo group. All furtheranalysis was confined to those subjects with a complete data set (Group1, n=11; Group 2, n=11; Group 3, n=10).

Study Design and Formulations

The normal subjects were divided into 3 groups of (initially) 12subjects and given the following supplements:

Group 1: L20; Z 2 mg/day

Group 2: MZ 10; L 10; Z 2 mg/day

Group 3: Placebo 0 mg/day

The carotenoid formulations were in 0.3 ml vegetable oil and wereadministered in soft gel capsules.

Visual performance was assessed as described in detail below, atbaseline, 3 months and at 6 months.

Statistical Analysis

The statistical software package PASW Statistics 18.0 (SPSS Inc.,Chicago, Ill., USA) was used for analysis. All quantitative variablesinvestigated exhibited a typical normal distribution. Means±SDs arepresented in the text and tables. Statistical comparisons of the threesupplementation groups, at baseline, were conducted using one way ANOVA,while paired samples t tests and repeated measures ANOVA (using ageneral linear model approach) were used to analyze visual performanceand MPOD measures in each supplementation group for change across studyvisits as appropriate. Where relevant, the Greenhouse-Geisser correctionfor violation of sphericity was used. A 5% level of significance wasused throughout the analysis.

Results

1. Baseline Analysis

Following randomization, one-way analysis of variance revealed nosignificant differences between groups at baseline, in terms ofdemographic, macular pigment, visual performance parameters, or otherparameters, as illustrated for selected parameters in table 14 below.

TABLE 14 Group 1: Group 2: Group 3: Variable Mean ± SD Mean ± SD PlaceboP value N 12 12 12 Age 56 ± 8  51 ± 13 46 ± 20 0.3 BMI 27 ± 3  25 ± 3 26 ± 5  0.31 BCVA 107 ± 5  109 ± 6  108 ± 6  0.72 MPOD 0.25 0.32 ± 0.130.37 ± 0.13 0.35 ± 0.18 0.69 MPOD 0.5 0.25 ± 0.14 0.27 ± 0.12 0.28 ±0.16 0.88 MPOD 1.0 0.15 ± 0.14 0.20 ± 0.07 0.16 ± 0.11 0.46 MPOD 1.750.07 ± 0.10 0.10 ± 0.07 0.04 ± 0.04 0.16 MPOD 3 0.07 ± 0.08 0.08 ± 0.070.04 ± 0.05 0.26 SD = standard deviation; BMI = body mass index; BCVA =best corrected visual acuity; MPOD = macular pigment optical density

2. MPOD Response at 3 and 6 Months

MPOD Measurement

A spatial profile of MPOD was generated across 0.25°, 0.5°, 1°, 1.75°and 3° of retinal eccentricity in relation to a 7° reference location,using the Macular Densitometer™, which employs a heterochromatic flickerphotometry (HFP) technique. Subjects were shown an explanatory video ofthe technique, and afforded a practice session prior to testcommencement. HFP flicker frequencies were optimized followingdetermination of individual critical flicker fusion (CFF) frequencymeasurements, in a customization process that optimizes MP measurements,(Stringham et al, Exp. Eye res. 2008, 87, 445-453). The MPOD measurementcomprised the average of six readings (computed as the radiance value atwhich the subject reported null flicker) at each retinal eccentricity,and was deemed reliable and acceptable only when the standard deviationof null flicker responses was below 0.1

TABLE 15 MPOD response and significance at each retinal eccentricityacross study visits T RM Group Intervention Baseline 3 months T test 6months Test ANOVA MPOD0.25 MPOD0.25 p* MPOD0.25 p** p*** 20 mg L; 2 mg Z0.32 ± 0.12 0.38 ± 0.15 0.080 0.41 ± 0.14 0.444 0.092 10 mg MZ; 10 mg L;2 mg Z 0.37 ± 0.13 0.49 ± 0.14 0.002 0.50 ± 0.20 0.012 0.002 Placebo0.35 ± 0.20 0.38 ± 0.20 0.709 0.37 ± 0.18 0.637 0.814 MPOD0.50 MPOD0.50p MPOD0.50 P p 20 mg L; 2 mg Z 0.27 ± 0.13 0.32 ± 0.22 0.456 0.30 ± 0.140.459 0.096 10 mg MZ; 10 mg L; 2 mg Z 0.28 ± 0.12 0.38 ± 0.16 0.011 0.37± 0.21 0.042 0.010 Placebo 0.28 ± 0.17 0.31 ± 0.16 0.404 0.28 ± 0.160.966 0.572 MPOD1.0 MPOD1.0 p MPOD1.0 P p 20 mg L; 2 mg Z 0.16 ± 0.140.18 ± 0.12 0.455 0.15 ± 0.14 0.767 0.533 10 mg MZ; 10 mg L; 2 mg Z 0.21± 0.08 0.28 ± 0.10 0.035 0.27 ± 0.14 0.085 0.047 Placebo 0.16 ± 0.120.14 ± 0.11 0.954 0.13 ± 0.10 0.400 0.997 MPOD1.75 MPOD1.75 p MPOD1.75 Pp 20 mg L; 2 mg Z 0.08 ± 0.10 0.08 ± 0.10 0.859 0.07 ± 0.10 0.867 0.92910 mg MZ; 10 mg L; 2 mg Z 0.11 ± 0.07 0.19 ± 0.05 0.005 0.18 ± 0.100.041 0.036 Placebo 0.03 ± 0.03 0.03 ± 0.05 0.767 0.03 ± 0.05 0.7320.815 MPOD3.0 MPOD3.0 p MPOD3.0 P p 20 mg L; 2 mg Z 0.05 ± 0.02 0.07 ±0.06 0.588 0.03 ± 0.03 0.185 0.671 10 mg MZ; 10 mg L; 2 mg Z 0.09 ± 0.070.11 ± 0.11 0.275 0.10 ± 0.07 0.707 0.915 Placebo 0.02 ± 0.03 0.02 ±0.03 0.810 0.02 ± 0.05 0.682 0.480 *difference between baseline and 3months (paired samples t test) **difference between baseline and 6months (paired samples t test) ***repeated measures ANOVA across allvisits

It can be seen here that the greatest increase in MP, at alleccentricities measured, can be seen in Group 2, a supplement containing10 mg MZ; 10 mg L; 2 mg Z.

Visual Performance Assessment

Visual acuity (VA) was measured at baseline with a computer-generatedlogMAR test chart (Test Chart 2000 Pro; Thompson Software Solutions,Hatfield, UK) at a viewing distance of 4 m, using the Sloan ETDRSletterset. VA was measured using a single letter scoring visual acuityrating, and recorded as the average of three measurements facilitated bythe software letter randomization feature. The eye with better visualacuity was chosen as the study eye; however, when both eyes had the samecorrected acuity, the right eye was chosen as the study eye.

Contrast sensitivity was measured using a functional acuity contrasttest (Optec6500 Vision Tester; Stereo Optical Co. Inc, Chicago, Ill.),which incorporates sine wave gratings, presented as Gabor patches, atspatial frequencies of 1.5, 3, 6, 12 and 18 cycles per degree (cpd) toproduce a contrast sensitivity function. Testing was performed undermesopic (3 candelas per square meter [cd/m²]) and photopic (85 cd/m2)conditions. (By way of explanation, 3 candelas per square meter isconsidered to represent the upper limit of mesopic conditions: anygreater level of illumination is considered to constitute photopicconditions). Contrast sensitivity testing was performed using a ThomsonChart or using the EDTRS (Early Treatment Diabetic Retinopathy Study)letters in log MAR form at five different spatial frequencies (seeLorente-Velazquez et al., 2011 Optom. Vis Sci. 88 (10): 1245-1251).

Glare disability was assessed using the same test, and testingconditions, but in the presence of an inbuilt circumferential LED glaresource (42 lux for mesopic and 84 lux for photopic glare testing). TheLED glare source rendered a daylight simulating color temperature of6500° K, and a spectral emission profile with a single large peak at 453nm (close to peak MP spectral absorbance). These tests have beendescribed in more detail elsewhere (Loughman et al. Vis Res. 2010;50:1249-1256; Nolan et al. Vis Res. 2011; 51:459-69). The subject task,and nature of the test were explained in detail prior to testcommencement, and subject performance was monitored closely by a trainedexaminer during the test, and reinstructed if necessary. Pupil diameterwas measured for the background mesopic and photopic conditions used,and also in the presence of both glare sources using a NeuropticsVIP™-200 pupillometer (Neuroptics Inc., Irvine, Calif. 92612, USA).

Photostress recovery time (PRT) of the short wavelength sensitive (SWS)visual system was assessed using a macular automated photostress (MAP)test, an adaptation of the Humphrey visual field analyzer (Model 745iCarl Zeiss Meditec Inc. Dublin, Calif., USA) for the assessment offoveal incremental light threshold (Dhalla et al., Am J Ophthalmol.2007; 143(4), 596-600). To isolate SWS cones, mid and long wavelengthsensitive cones were desensitized using a three minute sustainedexposure to a 100 cd/m², 570 nm bleaching background. A Goldmann V, 440nm stimulus, presented for 200 milliseconds, was used to test thesensitivity of the SWS system before and after photostress. Followingthe three minute adaptation and practice session (during which subjectperformance was assessed for reliability and understanding), subjectswere directed to fixate centrally between four circumferential lightstimuli, and to respond to the detection of a “blue” stimulus at thatlocation using the response button provided. Foveal sensitivity wasdetermined as the average of three consecutive measurements recorded indecibels (dB), with each dB representing a 0.1 log unit sensitivityvariation. Following baseline foveal sensitivity calculation, thesubject was exposed to a short wavelength dominated photostressstimulus, which consisted of a 5-s exposure to a 300-W lamp viewed at 1m through a low-pass glass dichroic filter, thus creating a temporaryfoveal “blue” after-image to mask fixation and reduce fovealsensitivity. Immediately post-photostress, a continuous and timed cycleof foveal sensitivity measurements were conducted and recorded. Thereduction in foveal sensitivity from baseline, along with the recoverycharacteristics of the SWS system sensitivity, was recorded. Pupildiameter was again recorded for background light conditions, and in thepresence of the photostress light source.

Ocular straylight was measured using an Oculus C-Quant (OCULUSOptikgeräte GmbH, Wetzlar, Germany), an instrument designed to quantifythe effect of light scatter on vision. A central bipartite 14° testfield was viewed monocularly through the instrument eyepiece. Subjectswere instructed to respond, using the appropriate response button, toindicate the position of the most strongly flickering right or left testhemi-field. Subjects were allowed a defined practice session, duringwhich reliable understanding of the task was assessed by the trainedexaminer. Test results were deemed acceptable only when the standarddeviation of measured straylight value (esd) was ≦0.08, and thereliability coefficient (Q) was ≧1. Absolute straylight values wererecorded in logarithmic form [log(s)].

Visual discomfort was assessed during the glare disability andphotostress testing procedures. Subjects were asked to rate theirdiscomfort immediately following presentation of the glare andphotostress light sources on a scale ranging from 1-10, where “1”indicated “no ocular discomfort”, “5” indicated “moderate oculardiscomfort”, and “10” indicated “unbearable ocular discomfort”. Such ascale has previously been used effectively in an exemplar macularpigment/glare study (Stringham et al., Invest Ophthalmol Vis Sci. 2011;52(10):7406-15). Visual experience was also assessed by questionnaire,using a modified version of the Visual Activities Questionnaire, as usedand described in detail elsewhere (Loughman et al. Vis Res. 2010;50:1249-1256; Sloane et al., The Visual Activities Questionnaire:Developing an instrument for assessing problems in everyday visualtasks. Technical Digest, Noninvasive Assessment of the Visual System,Topical Meeting of the Optical Society of America, January 1992). Iriscolor was also graded using a standardized iris classification scheme asdefined by Seddon et al. (Invest Ophthalmol Vis Sci 1990 (31),8:1592-1598).

3. BCVA demonstrated no significant effect for any of the interventiongroups at 3 months. At 6 months, pair t-test analysis revealed astatistically significant improvement in BCVA compared to baseline forgroup 2(p=0.008). Repeated measures ANOVA confirmed a significant changeacross the three study visits for group 2 (p=0.034).

4. Contrast Sensitivity

Mesopic and photopic contrast sensitivity improved from baseline valuesacross a range of spatial frequencies at three months, and inparticular, at six months. At three months, statistically significantimprovements were noted at 1.5 cpd (p=0.008) for mesopic conditions, andat 3 cpd (p=0.024) and 12 cpd (p=0.025) for photopic conditions forGroup 2. At six months, statistically significant improvements in CSwere noted across a substantially broader set of spatial frequencies,most notably under mesopic conditions, for Group 2, Mesopic CS at 6 CPDimproved significantly for Group 1 at 6 months (p<0.05). Repeatedmeasures ANOVA confirms the improvements in contrast sensitivity to bestatistically significant across all study visits for at least 3 of the5 spatial frequencies tested under mesopic and photopic conditions. Adetailed summary of contrast sensitivity results are provided in Table16.

TABLE 16 Contrast sensitivity change and significance levels at eachspatial frequency tested under mesopic and photopic conditions Contrastsensitivity Contrast sensitivity T Test RM ANOVA Group Intervention atbaseline at six months p* p** Photopic at 1.5 cpd Photopic at 1.5 cpd 20mg L; 2 mg Z 44 ± 26 53 ± 20 0.05 0.12 10 mg MZ; 10 mg L; 2 mg Z 49 ± 3068 ± 28 0.07 0.12 Placebo 52 ± 22 62 ± 29 0.41 0.28 Photopic at 3.0 cpdPhotopic at 3.0 cpd 20 mg L; 2 mg Z 85 ± 37 85 ± 29 0.96 0.68 10 mg MZ;10 mg L; 2 mg Z 73 ± 25 100 ± 28  0.002 0.002 Placebo 95 ± 36 94 ± 460.84 0.81 Photopic at 6.0 cpd Photopic at 6.0 cpd 20 mg L; 2 mg Z 99 ±27 100 ± 28  0.71 0.43 10 mg MZ; 10 mg L; 2 mg Z 95 ± 36 114 ± 45  0.230.26 Placebo 103 ± 54  116 ± 64  0.83 0.88 Photopic at 12.0 cpd Photopicat 12.0 cpd 20 mg L; 2 mg Z 30 ± 10 39 ± 17 0.178 0.26 10 mg MZ; 10 mgL; 2 mg Z 32 ± 13 50 ± 30 0.011 0.008 Placebo 57 ± 43 62 ± 42 0.643 0.92Photopic at 18.0 cpd Photopic at 18.0 cpd 20 mg L; 2 mg Z 8 ± 5 12 ± 9 0.168 0.38 10 mg MZ; 10 mg L; 2 mg Z 12 ± 6  23 ± 17 0.059 0.042 Placebo20 ± 17 17 ± 14 0.527 0.73 Mesopic at 1.5 cpd Mesopic at 1.5 cpd 20 mgL; 2 mg Z 57 ± 30 63 ± 23 0.618 0.83 10 mg MZ; 10 mg L; 2 mg Z 52 ± 1876 ± 24 0.003 0.000 Placebo 65 ± 27 75 ± 24 0.201 0.24 Mesopic at 3.0cpd Mesopic at 3.0 cpd 20 mg L; 2 mg Z 78 ± 45 74 ± 35 0.792 0.91 10 mgMZ; 10 mg L; 2 mg Z 58 ± 17 88 ± 38 0.003 0.001 Placebo 68 ± 39 96 ± 440.101 0.11 Mesopic at 6.0 cpd Mesopic at 6.0 cpd 20 mg L; 2 mg Z 41 ± 1353 ± 21 0.06 0.004 10 mg MZ; 10 mg L; 2 mg Z 50 ± 19 77 ± 49 0.14 0.058Placebo 53 ± 46 63 ± 43 0.58 0.82 Mesopic at 12.0 cpd Mesopic at 12.0cpd 20 mg L; 2 mg Z 7 ± 4 9 ± 6 0.198 0.16 10 mg MZ; 10 mg L; 2 mg Z 10± 6  33 ± 30 0.040 0.01 Placebo 13 ± 14 21 ± 25 0.400 0.50 Mesopic at18.0 cpd Mesopic at 18.0 cpd 20 mg L; 2 mg Z 2 ± 0 2 ± 0 NS 0.17 10 mgMZ; 10 mg L; 2 mg Z 2 ± 9 11 ± 14 0.047 0.021 Placebo 4 ± 5 5 ± 3 0.5930.28 RM ANOVA—Repeated measures ANOVA across all study visits; NS—nonsignificant (statistic not computed as SE of difference = 0) *differencebetween baseline and 6 months (paired samples t test) **repeatedmeasures ANOVA across all visits Group 1: n = 11; Group 2: n = 11; Group3: n = 10

5. Glare Disability

Mesopic and photopic glare disability improved from baseline across arange of spatial frequencies at three months and at six months. At threemonths, statistically significant improvements were noted at 12 cpd(p=0.048) for mesopic conditions, and at 1.5 cpd (p=0.023) and 3 cpd(p=0.033) for photopic conditions for Group 2. At six months,statistically significant improvements were noted across a substantiallybroader set of spatial frequencies for Group 2. Repeated measures ANOVAacross all study visits reveals no statistically significant change, atany spatial frequency, in mesopic or photopic glare disability forGroups 1 and 3. The statistically significant improvements in glaredisability for Group 2, under both mesopic and photopic conditions, forall spatial frequencies tested (other than 18 cpd) were robust torepeated measures ANOVA. A detailed summary of glare disability resultsare provided in Table 17.

TABLE 17 Glare disability change and significance levels at each spatialfrequency tested under mesopic and photopic conditions Glare DisabilityGlare Disability T test RM ANOVA Group Intervention at baseline at sixmonths p* p** Photopic at 1.5 cpd Photopic at 1.5 cpd Group 1: 20 mg L;2 mg Z 56 ± 27 67 ± 20 0.056 0.12 Group 2: 10 mg MZ; 10 mg L; 2 mg Z 50± 22 67 ± 22 0.059 0.033 Group 3: Placebo 60 ± 25 74 ± 29 0.134 0.24Photopic at 3.0 cpd Photopic at 3.0 cpd Group 1: 20 mg L; 2 mg Z 84 ± 2695 ± 31 0.175 0.28 Group 2: 10 mg MZ; 10 mg L; 2 mg Z 86 ± 24 121 ± 34 0.003 0.002 Group 3: Placebo 96 ± 30 97 ± 44 0.964 0.92 Photopic at 6.0cpd Photopic at 6.0 cpd Group 1: 20 mg L; 2 mg Z 114 ± 43  96 ± 37 0.1810.26 Group 2: 10 mg MZ; 10 mg L; 2 mg Z 91 ± 39 130 ± 40  0.032 0.04Group 3: Placebo 105 ± 51  112 ± 58  0.644 0.80 Photopic at 12.0 cpdPhotopic at 12.0 cpd Group 1: 20 mg L; 2 mg Z 34 ± 13 32 ± 14 0.785 0.13Group 2: 10 mg MZ; 10 mg L; 2 mg Z 42 ± 20 70 ± 25 0.004 0.006 Group 3:Placebo 29 ± 21 62 ± 48 0.06 0.13 Photopic at 18.0 cpd Photopic at 18.0cpd Group 1: 20 mg L; 2 mg Z 17 ± 11 23 ± 12 0.35 0.08 Group 2: 10 mgMZ; 10 mg L; 2 mg Z 33 ± 13 65 ± 20 0.17 0.23 Group 3: Placebo 33 ± 1546 ± 22 0.41 0.75 Mesopic at 1.5 cpd Mesopic at 1.5 cpd Group 1: 20 mgL; 2 mg Z 23 ± 8  45 ± 35 0.08 0.05 Group 2: 10 mg MZ; 10 mg L; 2 mg Z39 ± 26 58 ± 29 0.08 0.04 Group 3: Placebo 32 ± 24 38 ± 23 0.76 0.25Mesopic at 3.0 cpd Mesopic at 3.0 cpd Group 1: 20 mg L; 2 mg Z 36 ± 1061 ± 43 0.066 0.06 Group 2: 10 mg MZ; 10 mg L; 2 mg Z 40 ± 14 74 ± 400.009 0.02 Group 3: Placebo 54 ± 39 59 ± 46 0.820 0.93 Mesopic at 6 cpdMesopic at 6 cpd Group 1: 20 mg L; 2 mg Z 64 ± 41 90 ± 53 0.15 0.17Group 2: 10 mg MZ; 10 mg L; 2 mg Z 50 ± 19 77 ± 49 0.07 0.049 Group 3:Placebo 53 ± 46 64 ± 43 0.66 0.71 Mesopic at 12 cpd Mesopic at 12 cpdGroup 1: 20 mg L; 2 mg Z 5 ± 2 10 ± 17 0.303 0.35 Group 2: 10 mg MZ; 10mg L; 2 mg Z 5 ± 2 12 ± 8  0.016 0.014 Group 3: Placebo 7 ± 5 10 ± 7 0.238 0.15 Mesopic at 18 cpd Mesopic at 18 cpd Group 1: 20 mg L; 2 mg Z2 ± 0 2 ± 0 0.34 0.44 Group 2: 10 mg MZ; 10 mg L; 2 mg Z 2 ± 1 11 ± 130.16 0.21 Group 3: Placebo 4 ± 5 5 ± 3 0.14 0.22 cpd—Cycles per degree*difference between baseline and 6 months (paired samples t test)**repeated measures ANOVA across all visits Group 1: n = 11; Group 2: n= 11; Group 3: n = 10

6. Photostress Recovery Time

Photostress recovery time did not improve significantly for any of theGroups during the study period (p>0.05 for all). Paired t test analysisrevealed, however, that the improvement in PRT for Group 2 (PRT 37seconds [or 21%] shorter on average at six months compared to baseline)approached, but did not reach statistical significance (t=2.067,p=0.069).

Ocular straylight measures did not change significantly for any Group(p>0.05 for all). Visual experience and ocular discomfort, as determinedby questionnaire and discomfort rating, did not change significantlyduring the study period for any Group.

7. Comparison of Changes in MP with Changes in Visual PerformanceParameters

A comparison was made between changes in macular pigment and changes invisual performance parameters between baseline and 6 months. There wasno statistically significant relationship between change in macularpigment and any of the visual performance variables (p>0.05 for all).Table 18 gives the results for photopic (daytime) and mesopic(night-time) contrast sensitivity at 1.5 cpd.

TABLE 18 Changes in macular pigment (at 0.25° eccentricity) comparedwith changes in the following visual performance parameters betweenbaseline and 6 months: BCVA, photopic (daytime) contrast sensitivity,mesopic (night-time) contrast sensitivity, photopic contrast sensitivityunder glare conditions, mesopic contrast sensitivity under glareconditions. r p Change in MP vs change in BCVA Group 1 (20 L, 2 Z)−0.075 0.827 Group 2 (10 L, 10 MZ, 2 Z) 0.09 0.794 Group 3 (placebo)0.119 0.743 Change in MP vs change in photopic CS (1.5 cpd) Group 1 (20L, 2 Z) −0.104 0.76 Group 2 (10 L, 10 MZ, 2 Z) −0.154 0.651 Group 3(placebo) −0.242 0.5 Change in MP vs change in mesopic CS (1.5 cpd)Group 1 (20 L, 2 Z) 0.394 0.231 Group 2 (10 L, 10 MZ, 2 Z) −0.082 0.81Group 3 (placebo) −0.179 0.621 Change in MP vs change in photopic GD(1.5 cpd) Group 1 (20 L, 2 Z) −0.348 0.294 Group 2 (10 L, 10 MZ, 2 Z)0.263 0.435 Group 3 (placebo) 0.331 0.351 Change in MP vs change inmesopic GD (1.5 cpd) Group 1 (20 L, 2 Z) 0.394 0.231 Group 2 (10 L, 10MZ, 2 Z) −0.082 0.81 Group 3 (placebo) −0.179 0.621 Abbreviations: MP =macular pigment; BCVA = best corrected visual acuity; L = lutein; Z =zeaxanthin; MZ = meso-zeaxanthin; CS = contrast sensitivity; cpd =cycles per degree; GD = glare disability.

Surprisingly, these data show that the observed increases in visualperformance parameters were independent of the increases in macularpigment.

Discussion

In terms of MPOD, there was no significant change at any eccentricity,at 3 or at 6 months, in subjects supplemented with a preparation thatdoes not contain MZ or in subjects given placebo. In contrast, subjectssupplemented with all three macular carotenoids exhibited a significantincrease in MPOD at 4 of the 5 eccentricities tested, at 3 months and at6 months.

The current study demonstrates a novel and important effect of MPaugmentation on visual performance among healthy subjects without oculardisease. Across a broad range of testing modalities and conditions,visual performance improved significantly among subjects who exhibited asignificant rise in MPOD. Specifically, improvements in contrastsensitivity and glare disability (across virtually all spatialfrequencies, and under daytime and nighttime conditions), andimprovements in visual acuity, were demonstrated in subjects supplementwith all three macular carotenoids, but no such observations were seenin the placebo control subjects or in subjects supplemented with L and Z(but not MZ).

The data support the view that MP may influence visual performancethrough its optical filtration effects, as the glare disability testprotocol included an LED glare source that exhibited a short wavelengthpeak emission profile matching the known spectral absorbance of MP. Theobserved improvements in acuity and contrast sensitivity, however, areless consistent with a solely optical explanation. The stimuli used do,however, contain a relatively small short wavelength component. It ispossible, therefore, that MP augmentation results in optical imageenhancement through a reduction of the deleterious effects of chromaticaberration and light scatter, and thereby improves visual acuity andcontrast sensitivity, even for such spectrally broadband stimuli. It isalso possible that the macular carotenoids, which are intracellularcompounds, also play a neurobiological role, thereby contributing to,and/or facilitating, optimal neurophysiological performance, and hencevisual function (the limits of spatial vision represent the combinedinfluence of optical and neural efficiency limits). This view issupported by observation that there was no correlation between increasesin visual performance and increases in macular pigment, suggesting thatthe MP carotenoids may exert effects on visual performance by aneuro-physiological mechanism.

In conclusion, we have demonstrated a rapid and sustained rise in MPODfollowing supplementation with all three macular carotenoids, and thiswas not observed in placebo-controlled subjects or in subjectssupplemented with a preparation lacking MZ. Further, supplementationwith all three macular carotenoids resulted in significant improvementsin contrast sensitivity and glare disability (under photopic andscotopic conditions) and in corrected distance visual acuity, whereas nosuch changes were seen in placebo controls or in subjects supplementedwith a preparation lacking MZ. These findings have potentially importantimplications for people engaged in activities where optimization ofvisual importance is important (especially if operating under brightconditions), and warrant further study.

Example 5 Effect of a Supplement Containing MZ on Visual Performance inSubjects with an Atypical Distribution of Macular Pigment (a CentralDip)

Subjects and Dosage

Eight subjects with pre-identified central dips in their macular pigmentspatial profile as described in example 2 were recruited into thisstudy. All eight subjects consumed a supplement containing 10 mg L, 10mg MZ, and 10 mg Z daily for 3 months.

Methods

Macular pigment optical density (MPOD) was measured as in Example 1 atbaseline and after 3 months of MZ supplementation. Letter contrastsensitivity (Thomson Chart) was likewise measured using the methoddescribed in Example 3 section 4

Results

-   -   1. MPOD results: As seen from Table 19 and FIG. 10, the spatial        profile of MP was normalised following supplementation with 10        mg L, 10 mg MZ, and 10 mg Z for 3 months. All subjects responded        to this intervention. Statistically significant increases were        seen at all eccentricities except for 0.5°.

TABLE 19 Eccentricity Baseline 3 months p 0.25° 0.51 ± 0.25 0.64 ± 0.21<0.001 0.5°  0.54 ± 0.25 0.57 ± 0.20 0.140 1°   0.37 ± 0.20 0.43 ± 0.210.016 1.75° 0.20 ± 0.12 0.26 ± 0.12 0.008

-   -   2. Contrast sensitivity: as seen from Table 20 there was an        improvement in contrast sensitivity following supplementation        with 10 mg L, 10 mg MZ, and 10 mg Z for 3 months.

TABLE 20 Contrast sensitivity Baseline 3 months p 1.2 cpd 2.00 ± 0.152.07 ± 0.12 0.103 2.4 cpd 1.86 ± 0.16 2.02 ± 0.19 0.003 6 cpd 1.56 ±0.19 1.71 ± 0.21 <0.001 9.6 cpd 1.34 ± 0.21 1.46 ± 0.18 0.051 15.15 cpd1.02 ± 0.16 1.11 ± 0.20 0.035

Example 6

In one embodiment, the composition of the invention takes the form of amineral- and vitamin-containing dietary supplement, augmented with MZ, Land, optionally Z. The supplement is formulated as a tablet, with thefollowing composition of active ingredients:

MZ 5 mg L 5 mg Z 1 mg Vitamin A 800 micrograms Thiamin 1.1 mg Riboflavin1.4 mg Vitamin B6 2.0 mg Vitamin B12 2.5 micrograms Folic acid 400micrograms Niacin 20 mg Pantothenic Acid 6 mg Biotin 50 microgramsVitamin C 80 mg Vitamin D 20 micrograms Vitamin E 12 mg Calcium 120 mgMagnesium 60 mg Iron 14 mg Zinc 10 mg Copper 1 mg Iodine 150 microgramsManganese 3 mg Chromium 40 micrograms Selenium 55 micrograms Molybdenum50 micrograms

The following ingredients may be used as a source of the minerals andvitamins.

Minerals: calcium carbonate, magnesium hydroxide, ferrous fumarate, zincoxide, copper sulphate, potassium iodide, manganese sulphate, chromicchloride, sodium selenate, sodium molybdate

Vitamins: Retinyl acetate, Thiamin mono nitrate, Riboflavin, Pyridoxinhydrochloride, Cyano cobalomin, Folic Acid, Niacin,Calciun-D-pantothenate, D-biotin, Sodium Ascorbate#, Cholecalciferol,D-alpha-tocopherol acetate

The tablets may conveniently additionally comprise one or more of thefollowing fillers: Malto dextrin, Microcellulose, Hydroxy propyl methylcellulose, Shellac, Talcum, Gum acacia, Glycerol, Titanium dioxide,Polyfructose

One tablet (e.g. 500 mg) to be taken per day.

Example 7 Provision of MZ in Egg Yolks for Human Consumption

Several workers have shown that uptake of L and Z from egg yolks is 2-4times more efficient than from capsules (Handleman et al, 1999 Am. J.Clin. Nutr. 70, 247-251; Goodrow et al., 2006 J. Nutr. 136, 2519-2524;Johnson 2004, J. Nutr. 134, 1887-1893).

The objective of this study was to feed hens a mixture of L, MZ and Z todetermine the total amount of MZ in the yolk. In addition 24 eggscollected at the end of the experiment were consumed by one subject, oneegg/day and the blood MZ composition determined.

Methods

Eight Bovan Goldline hens were obtained at approximately 18 weeks ofage.

When the hens were producing at least 8 eggs per day in total, the henswere isolated and fed only a commercial meal. The experiment was started1 week later when a premix containing the mixed carotenoids was added tothe meal. The premix provided 250 mg MZ/kg feed with proportions of L50, MZ 30, Z 20.

The yolk carotenoids were measured in mixtures prepared from all eggscollected at baseline, three and six weeks.

Preparation of Egg-Yolk Suspensions

Yolks were individually weighed and mixed with phosphate-buffered salineand made up to 50 ml. Two ml of each suspension was mixed in a separateuniversal tube for each of the three batches separately and stored at−40 C.

Carotenoid Extraction

(i) Egg Yolk Suspensions

The egg yolk suspension (0.1 ml) was mixed with 0.15 ml aqueous KOH (25gl100 mlwater), 0.15 ml absolute ethyl alcohol and 0.1 ml echinenone(internal standard, 0.4 mg/500 ml ethyl alcohol) in a glass extractiontube and incubated at 45 C for 45 minutes.

Solutions were then cooled and mixed vigorously with 1.5 ml hexane(containing BHT500 mg/l) and centrifuged to separate the hexane andaqueous layers. One ml of the upper hexane layer was transferred to anevaporating tube and the residue was re-extracted with 1.5 ml hexane.After centrifuging, 1.5 ml of the upper layer was removed and theextracts combined and evaporated to dryness under nitrogen at 40 C. Theresidue was made up to 0.15 ml with mobile phase (see Ultracarb HPLCbelow) and 0.1 ml was injected onto a Ultra Carb Column for HLPCanalysis.

(ii) Plasma

Blood (10 ml) from a human subject was collected in lithium heparintubes at baseline, day 12 and day 24 after consumption of one egg perday and centrifuged to provide plasma subsequently stored at −40 C.Plasma, 0.25 ml was mixed with 0.2 ml sodium dodecyl sulphate, 0.4 mlethyl acetate (internal standard). Hexane containing BHT (1.0 ml) wasadded and the mixture extracted vigorously for 4 minutes, centrifugedfor 10 mins and 0.7 ml of the upper hexane layer removed and evaporatedto dryness.

The residue was made up to 0.1 ml with mobile phase (see HPLC procedurebelow) and 0.05 ml was injected onto the column.

Liquid Chromatography (HPLC) to Measure L, MZ, Z

Separation and quantitation of the MZ was achieved using a two columnprocedure.

Ultracarb procedure: Extracts prepared as described above werereconstituted in a mobile phase comprising acetonitrile:methanol (85:15containing 0.1% triethylamine). Using the same solvent mixture at 1.5ml/min, extracts were chromatographed isocratically using a 3 micro mUltracarb ODS column (250×4.6 mm, Phenomenex, UK) and detected using aphotodiode-array detector (model 2996, Waters Ltd) to quantify L andZ+MZ at 450 nm. Eluent that coincided with the emergence of MZ+Z wascollected from the waste line and evaporated to dryness under nitrogen.

Chiral chromoatography: The Z+MZ extract was then reconstituted in 0.1ml of hexane:isopropanol (90:10) and 50 uL was chromatographed on a 10micro m Chiralpak® AD column (250×4.6 mm; Chiral Technologies Europe,67404 Illkirch Cedex, France) to determine the proportion of MZ and Zisomers using gradient elution at 0.8 ml/min starting with 90% hexaneand 10% isopropyl alcohol and increasing to 95% hexane in a lineargradient over 30 minutes.

Results

MZ in Egg Yolks

Mean (SD) weights of the yolks at baseline and at the end of weeks 3 and6 were 12.29 (0.35), 14.23 (0.87) and 15.73 (0.72) g respectively. TheMZ contents of the yolks are shown in Table 21. At baseline only L and Zwere present;

Feeding 250 ppm of the carotenoid mixture for 3 weeks produced egg yolkscontaining 2.78 mg MZ/yolk of which L was circa 76% Z 13% and MZ 11%.There was no further increase at 6 weeks

Plasma

The MZ content in plasma from one human subject consuming one egg perday are shown in table 22.

Baseline total MZ concentration was 0.81 micro mol/liter of which L was53% Z was 47% and MZ 0%. The concentration of L had almost trebled atday 12 but the concentration then fell to only double the baseline valueat day 24.

The increase in MZ+Z at days 12 and 24 was 30% and 23% respectively andwas due solely due to increase in MZ.

Conclusions

Feeding a mixture of carotenoids to chickens for 3 and 6 weeks increasedL+MZ+Z in the egg yolks and in plasma in a subject consuming one egg perday.

The MZ content per yolk was raised from circa 0.8 mg to 2.8 mg. Since itis known that the absorption of L and Z from egg yolk is enhanced, twoor three eggs from chickens fed a mixture of L, Z and MZ could providesufficient MZ to improve visual performance in the subject, althoughthis was not tested.

TABLE 21 MZ contents of egg yolks from chicken fed 250 mg/kg mixedcarotenoids micro grams per yolk Weeks L Z MZ Total 0 563 278 0 841 32100 366 315 2781 6 2260 328 272 2860

TABLE 22 MZ contents of plasma in one person consuming one egg per day(units are micro moles per litre). Day L Z MZ Total 0 0.55 0.26 0 0.8112 1.20 0.28 0.06 1.54 24 1.06 0.25 0.07 1.38

Example 8 The Addition of MZ to Dietary Formulations and VP

A dry powder formula dietary supplement composition can be prepared bymixing 5 mg MZ, 5 mg L and 1 mg Z with the contents of 4 sachetscontaining circa 50 g each of “The Cambridge Diet” product, obtainedfrom Cambridge Nutritional Foods Limited, Stafford House, Brakey Road,Corby NN17 5LU, United Kingdom (The Cambridge Diet is a registered trademark).

Example 9 Fish Oils, MZ and VP

The retina contain a high concentration of Omega 3 fatty acids which areespecially abundant in fish oils, for example oils from salmon, herring,mackerel, anchovies, sardines; also from krill and green-lipped muscles.Omega 3 fatty acids are found as eicosapentanoic acid C22.6n−3 (EPA) anddocosahexanoic acid C22.6n−3 (DHA) and combined make up about 30% offish body oil. The acceptable daily macro nutrient dose (AMND) ofEPA+DHA is about 1.6 g/day for men and 1.1 g/day for women, i.e. about 5g and 3.5 g fish oil respectively.

The occurrence of a high concentration of omega 3 fatty acids in theretina suggests that they may play and important role in vision. Acombination of macular carotenoids (MC) containing MZ with omega3 fattyacids would thus be beneficial to the retina and improve visualperformance. The mixture can be in capsules or as an emulsion in asachet. The latter has the advantage that fewer doses can be given in asachet whilst several large capsules (which many elderly people finddifficult to swallow) are needed for the AMND. The emulsion can containfrom 25-60% fish oils to provide from 0.5-2.0 g omega-3 fatty acids andsufficient MC to give a daily dose of 0.5 mg to 50 mg MC per day.

A commercial preparation of active macular carotenoids (MC) consistingof mesozeazanthin 10 g lutein 10 g and zeaxanthin 2 g in 78 ml krill oilis mixed with 900 ml salmon oil and made into soft gel capsules eachcontaining 1 g oil formulation. A daily dose of 5 capsules will providethe 1.5 g of Omega 3 fatty acids and a 22 mg dose of macular carotenoidsto improve visual performance.

An alternative embodiment may be formulated as follows:

Ingredients The mixture of and MC, krill and salmon oils as above 55%Water 35% Sucralose (Splenda ™) 4% Milk powder 5% Potassium sorbate 0.1%Alpha tocopherol 0.1% Flavorings (e.g. citrus) 0.8%

An emulsion is made under an inert atmosphere using standard techniquesand then packed into airtight sachets each containing 5 grams emulsion.The daily dose is 2 sachets per day containing 6 g omega 3 fatty acidsand 22 mg MC.

The invention claimed is:
 1. A capsule consisting essentially ofisolated mesozeaxanthin, isolated lutein, isolated zeaxanthin, fish oilcontaining an omega-3 fatty acid and Vitamin E.